4-heteroaryl-3-heteroarylidenyl-2-indolinones and their use as protein kinase inhibitors

ABSTRACT

The present invention relates to certain 4-heteroaryl-3-heteroarylidenyl-2-indolinones compounds and their physiologically acceptable salts which modulate the activity of protein kinases (“PKs”), in particular CDK2. The compounds of the present invention are therefore useful in treating disorders related to abnormal PK activity. Pharmaceutical composition containing these compounds and methods of preparing these compounds are also described.

CROSS-REFERENCE

This application claims priority under 35 U.S.C. 119(e) to provisionalapplication Serial No. 60/215,654, filed on Jun. 30, 2000, thedisclosure of which is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to certain4-heteroaryl-3-heteroarylidenyl-2-indolinones compounds and theirphysiologically acceptable salts which modulate the activity of proteinkinases (“PKs”). The compounds of the present invention are thereforeuseful in treating disorders related to abnormal PK activity.Pharmaceutical composition containing these compounds and methods ofpreparing these compounds are also described.

2. State of the Art

The following is offered as background information only and is notadmitted to be prior art to the present invention.

PKs are enzymes that catalyze the phosphorylation of hydroxy groups ontyrosine, serine and threonine residues of proteins. The consequences ofthis seemingly simple activity are staggering; cell growth,differentiation and proliferation, i.e., virtually all aspects of celllife in one way or another depend on PK activity. Furthermore, abnormalPK activity has been related to a host of disorders, ranging fromrelatively non life threatening diseases such as psoriasis to extremelyvirulent diseases such as glioblastoma (brain cancer) (see U.S. Pat. No.5,792,783 which is incorporated herein by reference in its entirety).

In view of the apparent link between PK-related cellular activities andwide variety of human disorders, a great deal of effort is beingexpended in an attempt to identify ways to modulate PK activity. Some ofthis effort has involved biomimetic approaches using large moleculespatterned on those involved in the actual cellular processes (e.g.,mutant ligands (U.S. Pat. No. 4,966,849); soluble receptors andantibodies (Published PCT Appl. WO 94/10202, Kendall and Thomas, Proc.Nat'l Acad. Sci., 90:10705-09 (1994), Kim, et al., Nature, 362:841-844(1993)); RNA ligands (Jelinek, et al., Biochemistry, 33:10450-56);Takano, et al., Mol. Bio. Cell 4:358A (1993); Kinsella, et al., Exp.Cell Res. 199:56-62 (1992); Wright, et al., J. Cellular Phys.,152:448-57) and tyrosine kinase inhibitors (Published PCT Appls. WO94/03427; 1 WO 92/21660; WO 91/15495; WO 94/14808; U.S. Pat. No.5,330,992; Mariani, et al., Proc. Am. Assoc. Cancer Res., 35:2268(1994)).

In addition to the above, attempts have been made to identify smallmolecules which act as PK inhibitors. For example, bis-monocylic,bicyclic and heterocyclic aryl compounds (Published PCT Appl. WO92/20642), vinyleneazaindole derivatives (Published PCT Appl. WO94/14808) and 1-cyclopropyl-4-pyridylquinolones (U.S. Pat. No.5,330,992) have been described as tyrosine kinase inhibitors. Styrylcompounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridylcompounds (U.S. Pat. No. 5,302,606), quinazoline derivatives (EP App.No.0 566 266 A1), selenaindoles and selenides (Published PCT Appl. WO94/03427), tricyclic polyhydroxylic compounds (Published PCT Appl. WO92/21660), benzylphosphonic acid compounds (Published PCT Appl. WO91/15495) and indolinone compounds (U.S. Pat. No. 5,792,783) have allbeen described as PTK inhibitors useful in the treatment of cancer.However these compounds have limited utility because of toxicity or poorbioavailability. Accordingly, there is a need for compounds thatovercome these limitations. The compounds of the present inventionfulfil this need.

SUMMARY OF THE INVENTION

In one aspect, this invention is directed to a compound of formula (I):

wherein:

R¹ and R² are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo,—CX₃, hydroxy, alkoxy, nitro, cyano, —C(O)R²⁶, —C(O)OR²⁶, R²⁶C(O)O—,—C(O)NR²⁸R²⁹, R²⁶C(O)NR²⁸—, —NR²⁸R²⁹,

—S(O)₂R²⁶, —S(O)₂OR²⁶, —S(O)₂NR²⁸R²⁹, R²⁶S(O)₂NR²⁸, X₃CS(O)₂— andX₃CS(O)₂NR²⁸— where X is F, Cl, Br, or I;

Het is selected from the group consisting of:

wherein:

A¹, A², A³, A⁴, and A⁵ are selected from the group consisting of carbonand nitrogen with the proviso that at least one and no more than two ofA¹, A², A³, A⁴, and A⁵ are nitrogen;

R³, R⁴, R⁵, R⁶ and R⁷ are independently selected from the groupconsisting of hydrogen, alkyl, halo, hydroxy, alkoxy, X₃C—, nitro,cyano, —NR²⁸R²⁹, —C(O)OR and —C(O)NR²⁸R²⁹ where X is as defined above;it being understood that when A¹, A², A³, A⁴ or A⁵ is nitrogen, R³, R⁴,R⁵, R⁶ or R⁷, respectively, does not exist;

D is carbon or nitrogen;

R⁸, R⁹, R¹¹ and R¹² are independently selected from the group consistingof hydrogen, alkyl, hydroxy, alkoxy, halo, nitro, cyano and —NR²⁸R²⁹;

Z is selected from the group consisting of oxygen, sulfur, and —NR¹⁰;

R¹⁰ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl, —C(O)R²⁶, —C(S)R²⁶, —C(O)OR²⁶, —C(O)NR²⁸R²⁹,—C(S)NR²⁸R²⁹, —C(NH)NR²⁸R²⁹ and —S(O)₂R²⁶;

E¹, E², E³ and E⁴ are selected from the group consisting of carbon,nitrogen, oxygen and sulfur with the proviso that when D is carbon thenat least one of E¹, E², E³ and E⁴ is other than carbon and that no morethan one of E¹, E², E³ or E⁴ is oxygen or sulfur;

the dotted circle inside the five-member ring contain D, E¹, E², E³ andE⁴ ring means that the ring system is aromatic;

R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently selected from the groupconsisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl,heteroalicyclic, hydroxy, alkoxy, mercapto, thioalkoxy, halo, nitro,cyano, —C(O)R²⁶, —C(O)OR²⁶, —C(O)NR²⁸R²⁹ and —NR²⁸R²⁹, it beingunderstood that, when one of E¹, E², E³ or E⁴ is sulfur or oxygen andany of the others is nitrogen, there is no R group bonded to any ofthose nitrogens, it also being understood that, when two or three of E¹,E², E³ or E⁴ are nitrogen, there is an R group bonded to one of thenitrogens and that R group is selected from the group consisting ofhydrogen and alkyl, there being no R group bonded to any of the othernitrogens;

Q is selected from the group consisting of:

where:

G¹, G², G³, G⁴ and G⁵ are selected from the group consisting of carbonand nitrogen with the proviso that no more than two of G¹, G², G³, G⁴and G⁵ are nitrogen;

R¹⁷, R¹⁸, R¹⁹, R²⁰ and R²¹ are independently selected from the groupconsisting of hydrogen, alkyl, hydroxy, alkoxy, halo,

—NR²⁸R²⁹, —(CH₂)_(n)C(O)R²⁶, —(CH₂)_(n)C(O)OR²⁶ and—(CH₂)_(n)C(O)NR²⁸R²⁹,

—(CH₂)_(n)NR²⁸R²⁹, —(CH₂)_(n)S(O)₂R²⁶ and —(CH₂)_(n)S(O)₂NR²⁸R²⁹;

J¹ is selected from the group consisting of nitrogen, oxygen and sulfursuch that when J¹ is nitrogen, R²² is selected from the group consistingof hydrogen, alkyl and —C(O)R²⁶; and

when J¹ is oxygen or sulfur, R²² does not exist;

J², J³ and J⁴ are selected from the group consisting of carbon andnitrogen;

R²³, R²⁴ and R²⁵ are independently selected from the group consisting ofhydrogen, alkyl, aryl optionally substituted with one or more groupsindependently selected from the group consisting of hydroxy,unsubstituted lower alkoxy and halo, halo,

—(CH₂)_(n)C(O)R²⁶, —(CH₂)_(n)C(O)OR²⁶ and —(CH₂)_(n)C(O)NR²⁸R²⁹,—(CH₂)_(n)NR²⁸R²⁹,

—(CH₂)_(n)S(O)₂R²⁶, —(CH₂)_(n)S(O)₂NR²⁸R²⁹, —(CH₂)_(n)OR²⁶,—O(CH₂)_(n)NR²⁸R²⁹ and —C(O)NH (CH₂)_(n)NR²⁸R²⁹;

n is 0, 1, 2, or 3;

R²³ and R²⁴ or R²⁴ and R²⁵ may combine to form a group selected from thegroup consisting of —CH₂CH₂CH₂CH₂—, —CH═CR³³—CR³⁴═CH— and

—C(O)Y(CH₂)₂— and group wherein Y is selected from the group consistingof oxygen, sulfur and —N(R²⁷)— and R³³ and R³⁴ are selected from thegroup consisting of hydrogen, —(CH₂)_(n)NR²⁸R²⁹ and —O(CH₂)_(n)NR²⁸R²⁹where, when one of R³³ or R³⁴ is —(CH₂)_(n)NR²⁸R²⁹ or

—O(CH₂)_(n)NR²⁸R²⁹, the other is hydrogen;

it being understood that, when J², J³ or J⁴ is nitrogen, R²³, R²⁴ orR^(25,) respectively, does not exist;

R²⁶ is selected from the group consisting of hydrogen, alkyl,cycloalkyl, aryl and heteroaryl;

R²⁷ is selected from the group consisting of hydrogen and alkyl;

R²⁸ and R²⁹ are independently selected from the group consisting ofhydrogen, alkyl, aryl, heteroaryl, —(CH₂)_(n)aryl, —(CH₂)_(n)heteroaryland —C(O)R²⁶, or, combined, R²⁸ and R²⁹ may form a group selected fromthe group consisting of —(CH₂)₅—, —(CH₂)₂O(CH₂)₂—, —(CH₂)₂NR³ (CH₂)₂—and —(CH)₃C(O)— wherein R³⁰ is selected from the group consisting ofhydrogen, alkyl, —C(O)R²⁶, —S(O)₂R²⁶, —S(O)₃R²⁶, —S(O)₂NR³¹R³²,—C(O)NHNR³¹R³², —C(O)NR³¹R³², —C(S)NR³¹R³² and —C(O)OR²⁶ where R³¹ andR³² are independently selected from the group consisting of hydrogen,unsubstituted lower alkyl and aryl optionally substituted with one ormore groups independently selected from the group consisting of halo andunsubstituted lower alkoxy; or a pharmaceutically acceptable saltthereof; provided that: the compound of formula (I) is not:

(Z)-1,3-dihydro-3-[(1H-pyrrol-2-yl)methylene]-4-(2-thiophenyl)-2H-indol-2-one;and

Z)-1,3-dihydro-4-(2,4-dimethoxy-6-pyrimidinyl)-3-[(1H-pyrrol-2-yl)methylene]-2H-indol-2-one.

In a second aspect, this invention relates to a method for themodulation of the catalytic activity of a PK by contacting a PK with acompound of this invention or a pharmaceutically acceptable saltthereof. The modulation of the catalytic activity of PKs using acompound of this invention may be carried out in vitro or in vivo.

The protein kinase whose catalytic activity is being modulated by acompound of this invention is selected from the group consisting ofreceptor protein tyrosine kinases, cellular (or non-receptor) tyrosinekinases and serine-threonine kinases.

Preferably, the receptor protein kinase whose catalytic activity ismodulated by a compound of this invention is selected from the groupconsisting of EGF, HER2,HER3,HER4, IR, IGF-1R, IRR, PDGFRα, PDGFRβ,CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R, FGFR-2R,FGFR-3R and FGFR-4R. The cellular tyrosine kinase whose catalyticactivity is modulated by a compound of this invention is selected fromthe group consisting of Src, Frk, Btk, Csk, Abl, ZAP70, Fes/Fps, Fak,Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk. Theserine-threonine protein kinase whose catalytic activity is modulated bya compound of this invention is selected from the group consisting ofCDK2 and Raf.

In a third aspect, this invention is directed to a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof formula (I) or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier or excipient. Such pharmaceuticalcomposition may contain both carriers and excipients as well as othercomponents generally known to those skilled in the formulation ofpharmaceutical compositions.

In a fourth aspect, this invention is directed to a method for treatingor preventing a protein kinase related disorder in an organism whichmethod comprises administering to said organism a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof formula (I) or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier or excipient.

The above-referenced protein kinase related disorders are those mediatedby receptor protein tyrosine kinases, non-receptor or cellular tyrosinekinases, and serine-threonine kinases.

Preferably, the protein kinase related disorders are those mediated byEGFR, a PDGFR, IGFR, flk (VEGFR), CDK2, Met kinase, and Src kinase.

More preferably, the disorders are cancer selected from the groupconsisting of squamous cell carcinoma, sarcomas such as Kaposi'ssarcoma, astrocytoma, glioblastoma, lung cancer, bladder cancer,colorectal cancer, gastrointestinal cancer, head and neck cancer,melanoma, ovarian cancer, prostate cancer, breast cancer, small-celllung cancer, glioma, colorectal cancer, genitourinary cancer andgastrointestinal cancer; diabetic retinopathy, a hyperproliferationdisorder, von Hippel-Lindau disease, restenosis, fibrosis, psoriasis,inflammatory disorders such as rheumatoid arthritis, osteoarthritis,immunological disorders such as autoimmune diseases, cardiovasulardisorders such as atherosclerosis and angiogenesis related disorders.

In a fifth aspect, this invention is directed to a use of a compound offormula (I) as a reference compound in an assay in order to identify newcompounds (test compounds) that modulate protein kinase activity whichmethod comprises contacting cells expressing said protein kinase with atest compound or a compound of formula (I) and then monitoring saidcells for an effect.

The above-referenced effect is selected from a change or an absence ofchange in a cell phenotype, a change or absence of change in thecatalytic activity of said protein kinase or a change or absence ofchange in the interaction of said protein kinase with a natural bindingpartner.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

The following terms used in the claims and the specification have themeanings given below. Other terms have their art recognized meaning.

The term “alkyl” refers to a saturated aliphatic hydrocarbon includingstraight chain and branched chain groups. Preferably, the alkyl grouphas 1 to 20 carbon atoms (whenever a numerical range; e.g. “1-20”, isstated herein, it means that the group, in this case the alkyl group,may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up toand including 20 carbon atoms). More preferably, it is a medium sizealkyl radical having 1 to 10 carbon atoms e.g., methyl, ethyl, propyl,2-propyl, n-butyl, iso-butyl, tert-butyl, pentyl, and the like. Mostpreferably, it is lower alkyl having 1 to 4 carbon atoms e.g., methyl,ethyl, propyl, 2-propyl, n-butyl, iso-butyl, or tert-butyl, and thelike.

The alkyl group may be substituted or unsubstituted. When substituted,the substituent group(s) is preferably one, two, or three, morepreferably one or two groups, independently selected from the groupconsisting of halo, hydroxy, unsubstituted lower alkoxy, mercapto,(unsubstituted lower alkyl)thio, cyano, nitro,

—C(O)R³³, —C(S)R³³, —OC(O)NR³⁴R³⁵, R³³OC(O)NR³⁴—, —C(S)NR³⁴R³⁵,R³³OC(S)NR³⁴—, —C(O)NR³⁴R³⁵, R³³C(O)NR³⁴—, R³³S(O)₂NR³⁴—, —S(O)₂NR³⁴R³⁵R³³ S(O)—, R³³S(O)₂—, —C(O)OR³³, R³³C(O)O—, —NR³⁴R³, aryl optionallysubstituted with one or more, more preferably one, two, or three groupsindependently selected from the group consisting of halo, hydroxy andunsubstituted lower alkoxy, aryloxy optionally substituted with one ormore, more preferably one, two, or three groups independently selectedfrom the group consisting of halo, hydroxy and unsubstituted loweralkoxy, arylthio optionally substituted with one or more, morepreferably one, two, or three groups independently selected from thegroup consisting of halo, hydroxy and unsubstituted lower alkoxy,6-member heteroaryl having from 1 to 3 nitrogen atoms in the ring, thecarbons in the ring being optionally substituted with one or more, morepreferably one or two groups independently selected from the groupconsisting of halo, hydroxy and unsubstituted lower alkoxy, 5-memberheteroaryl having from 1 to 3 heteroatoms in the ring selected from thegroup consisting of nitrogen, oxygen and sulfur, the carbon atoms of thegroup being optionally substituted with one or more, more preferably oneor two groups independently selected from the group consisting of halo,hydroxy and unsubstituted lower alkoxy groups and a 5- or 6-memberheteroalicyclic group having from 1 to 3 heteroatoms in the ringselected from the group consisting of nitrogen, oxygen and sulfur, thecarbon atoms in the group being optionally substituted with one or more,more preferably one or two groups independently selected from the groupconsisting of halo, hydroxy and unsubstituted lower alkoxy groups,wherein R³³ is selected from the group consisting of hydrogen,unsubstituted lower alkyl and aryl optionally substituted with one ormore, more preferably one, two, or three groups independently selectedfrom the group consisting of halo and unsubstituted lower alkoxy and R³⁴and R³⁵ are independently selected from the group consisting ofhydrogen, unsubstituted lower alkyl, —C(O)R³³, aryl optionallysubstituted with one, two, or three groups independently selected fromthe group consisting of halo and unsubstituted lower alkoxy andheteroaryl optionally substituted with one, two, or three groupsindependently selected from the group consisting of halo andunsubstituted lower alkoxy.

A “cycloalkyl” group refers to a 3 to 8 member all-carbon monocyclicring, an all-carbon 5-member/6-member or 6-member/6-member fusedbicyclic ring or a multicyclic fused ring (a “fused” ring system meansthat each ring in the system shares an adjacent pair of carbon atomswith each other ring in the system) group wherein one or more of therings may contain one or more double bonds but none of the rings has acompletely conjugated pi-electron system. Examples, without limitation,of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane,cyclopentene, cyclohexane, cyclohexadiene, adamantane, cycloheptane and,cycloheptatriene. A cycloalkyl group may be substituted orunsubstituted. When substituted, the substituent group(s) is preferablyone or more independently selected from the group consisting of halo,hydroxy, unsubstituted lower alkoxy, mercapto, (unsubstituted loweralkyl)thio, cyano, nitro, —C(O)R³³—C(S)R³³, —OC(O)NR³⁴R³⁵,R³³OC(O)NR³⁴—, —OC(S)NR³⁴R³⁵, R³³OC(S)NR³⁴—, —C(O)NR³⁴R³⁵, R³³C(O)NR³⁴—,R³³S(O)₂NR³⁴—, —S(O)₂NR³⁴R³⁵, R³³S(O)—, R³³S(O)₂—, —C(O)OR³³, R³³C(O)O—,—NR³⁴R³⁵, aryl optionally substituted with one, two or three groupsindependently selected from the group consisting of halo, hydroxy andunsubstituted lower alkoxy, aryloxy optionally substituted with withone, two or three groups independently selected from the groupconsisting of halo, hydroxy and unsubstituted lower alkoxy, arylthiooptionally substituted with with one, two or three groups independentlyselected from the group consisting of halo, hydroxy and unsubstitutedlower alkoxy, 6-member heteroaryl having from 1 to 3 nitrogen atoms inthe ring, the carbons in the ring being optionally substituted with withone, two or three groups independently selected from the groupconsisting of halo, hydroxy and unsubstituted lower alkoxy, 5-memberheteroaryl having from 1 to 3 heteroatoms in the ring selected from thegroup consisting of nitrogen, oxygen and sulfur, the carbon atoms of thegroup being optionally substituted with one or two groups independentlyselected from the group consisting of halo, hydroxy and unsubstitutedlower alkoxy groups and a 5- or 6-member heteroalicyclic group havingfrom 1 to 3 heteroatoms in the ring selected from the group consistingof nitrogen, oxygen and sulfur, the carbon atoms in the group beingoptionally substituted with with one, two or three groups independentlyselected from the group consisting of halo, hydroxy and unsubstitutedlower alkoxy groups, wherein R³³ is selected from the group consistingof hydrogen, unsubstituted lower alkyl and aryl optionally substitutedwith one, two or three groups independently selected from the groupconsisting of halo and unsubstituted lower alkoxy and R³⁴ and R³⁵ areindependently selected from the group consisting of hydrogen,unsubstituted lower alkyl, —C(O)R³³, aryl optionally substituted withwith one, two or three groups independently selected from the groupconsisting of halo and unsubstituted lower alkoxy and heteroaryloptionally substituted with with one, two or three groups independentlyselected from the group consisting of halo and unsubstituted loweralkoxy.

An “alkenyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon—carbondouble bond e.g., ethenyl, propenyl, butenyl, and the like.

An “alkynyl” group refers to an alkyl group, as defined herein,consisting of at least two carbon atoms and at least one carbon—carbontriple bond e.g., ethynyl, propynyl, and the like.

An “aryl” group refers to an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups of 6 to 12 ring atoms and having a completely conjugatedpi-electron system. Examples, without limitation, of aryl groups arephenyl, naphthalenyl and anthracenyl. The aryl group may be substitutedor unsubstituted. When substituted, the substituted group(s) ispreferably one or more, more preferably one, two, or three,independently selected from the group consisting of unsubstituted loweralkyl, X₃C—, halo, hydroxy, unsubstituted lower alkoxy, mercapto,(unsubstituted lower alkyl)thio, cyano, nitro, —C(O) R³³, —C(S)R³³,—C(O)NR³R³⁵, R³³OC(O)NR³⁴—, —OC(S)NR³⁴R³⁵, R³³OC(S)NR³⁴—, —C(O)NR³⁴R³⁵,R³³C(O)NR³⁴—, R³³S(O)₂NR³⁴—, —S(O)₂NR³⁴R³⁵, R³³S(O)—, R³³S(O)₂—,—C(O)OR³³, R³³C(O)O— and —NR³⁴R³⁵ with R³³, R³⁴ and R³⁵ as definedabove.

As used herein, a “heteroaryl” group refers to a monocyclic or fusedaromatic ring (i.e., rings which share an adjacent pair of atoms)containing 5 to 10 ring atoms wherein one, two, three or four ring atomsare independently selected from the group consisting of nitrogen, oxygenand sulfur, the rest being carbon. Examples, without limitation, ofheteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole,isoxazole, thiazole, pyrazole, pyridine, pyrimidine, pyrazine,pyridazine, quinoline, isoquinoline, purine and carbazole. With regardto the five-member heteroaryl groups containing two or more nitrogensand no other hetero atoms in the ring, such as imidazole and triazole,one of the nitrogens in the ring may be bonded to an R group while theothers may not. This gives rise to isomeric structures such as thoseshown below for dimethylimidazoles and dimethyltriazoles:

All such isomers are within the scope of this invention. A heteroarylgroup may be substituted or unsubstituted. When substituted, thesubstituted group(s) is preferably one or more, more preferably one ortwo groups independently selected from the group consisting ofunsubstituted lower alkyl, X₃C—, halo, hydroxy, unsubstituted loweralkoxy, mercapto, (unsubstituted lower alkyl)thio, cyano, nitro,—C(O)R³³, —C(S)R³³, —OC(O)NR³⁴R³⁵, R³³OC(O)NR³⁴—, —OC(S)NR³⁴R³⁵,R³³OC(S)NR³⁴—, —C(O)NR³⁴R³⁵, R³³C(O)NR³⁴—, R³³S(O)₂NR³⁴—, —S(O)₂NR³⁴R³⁵,R³³S(O)—, R³³S(O)₂—, —C(O)OR³³, R³³C(O)O— and —NR³⁴R³⁵ with R³³, R³⁴ andR³⁵ as defined above.

A “heteroalicyclic” group refers to a monocyclic or fused ring of 5 to10 ring atoms wherein one, two, or three ring atoms are independentlyselected from the group consisting of nitrogen, oxygen and sulfur, therest being carbon. The rings may also have one or more double bonds.However, the rings do not have a completely conjugated pi-electronsystem. The heteroalicyclic ring may be substituted or unsubstituted.When substituted, the substituted group(s) is preferably one or more,more preferably one or two groups independently selected from the groupconsisting of unsubstituted lower alkyl, X₃C—, halo, hydroxy,unsubstituted lower alkoxy, mercapto, (unsubstituted lower alkyl)thio,cyano, nitro, —C(O)R³³, —C(S)R³³, —OC(O)NR³⁴R³⁵, R³³C(O)NR³—,—OC(S)NR³⁴R³⁵, R³³OC(S)NR³⁴—, —C(O)NR³⁴R³⁵, R³³C(O)NR —, R³³S(O)₂NR³⁴—,—S(O)₂NR³⁴R³⁵, R³³S(O)—, R³³S(O)₂—, —C(O)OR³³, R³³C(O)O— and —NR³⁴R³⁵with R³³, R³⁴ and R³⁵ as defined above.

A “hydroxy” group refers to an —OH group.

An “alkoxy” group refers to both an —O-(unsubstituted alkyl) and an—O-(unsubstituted cycloalkyl) group.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group,as defined herein.

A “mercapto” group refers to an —SH group.

A “alkylthio” group refers to both an —S(unsubstituted alkyl) and an—S(unsubstituted cycloalkyl) group.

A “thioalkoxy” group refers to both an S-alkyl and an —S-cycloalkylgroup, as defined herein.

A “arylthio” group refers to both an —S(aryl) and an —S(heteroarylgroup), as defined herein.

A “halo” group refers to fluorine, chlorine, bromine or iodine.

A “cyano” group refers to a —C≡N group.

A “nitro” group refers to a —NO₂ group.

“Heteroaryl” refers to both heteroaryl groups, defined elsewhere hereinand exemplified, without limitation, by the compounds of Group I, below,and heteroalicyclic groups, likewise defined elsewhere herein and, againwithout limitation, exemplified by the compounds of Group II, below:

As used herein “heteroarylidenyl” refers to a group having the followingstructure, wherein Q is a heteroaryl group, as defined above.

The terms “2-oxindole,” “2-indolinone,” and “indolin-2-one” are usedinterchangeable to refer to a group having the following structure. The3 and 4 positions, wherein compounds of this invention are substitutedwith a heteroarylidenyl or a heteroaryl group, respectively, are marked:

As used herein, the term “combined,” when referring to two R groupsbonded to adjacent carbon atoms, means that the atoms shown ascomprising the “combined” structure form a bridge from the carbon towhich one of the R groups is bonded to the carbon atom to which theother R group is bonded.

As used herein, “PK” refers to receptor protein tyrosine kinase (RTKs),non-receptor or “cellular” tyrosine kinase (CTKs) and serine-threoninekinases (STKs).

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures by,practitioners of the chemical, pharmaceutical, biological, biochemicaland medical arts.

As used herein, the term “modulation” or “modulating” refers to thealteration of the catalytic activity of RTKs, CTKs and STKs. Inparticular, modulating refers to the activation or inhibition of thecatalytic activity of RTKs, CTKs and STKs, preferably the activation orinhibition of the catalytic activity of RTKs, CTKs and STKs, dependingon the concentration of the compound or salt to which the RTK, CTK orSTK is exposed or, more preferably, the inhibition of the catalyticactivity of RTKs, CTKs and STKs.

The term “catalytic activity” as used herein refers to the rate ofphosphorylation of tyrosine under the influence, direct or indirect, ofRTKs and/or CTKs or the phosphorylation of serine and threonine underthe influence, direct or indirect, of STKs.

The term “contacting” as used herein refers to bringing a compound ofthis invention and a target PK together in such a manner that thecompound can affect the catalytic activity of the PK, either directly,i.e., by interacting with the kinase itself, or indirectly, i.e., byinteracting with another molecule on which the catalytic activity of thekinase is dependent. Such “contacting” can be accomplished “in vitro,”i.e., in a test tube, a petri dish or the like. In a test tube,contacting may involve only a compound and a PK of interest or it mayinvolve whole cells. Cells may also be maintained or grown in cellculture dishes and contacted with a compound in that environment. Inthis context, the ability of a particular compound to affect a PKrelated disorder, i.e., the IC₅₀ of the compound, defined below, can bedetermined before use of the compounds in vivo with more complex livingorganisms is attempted. For cells outside the organism, multiple methodsexist, and are well-known to those skilled in the art, to get the PKs incontact with the compounds including, but not limited to, direct cellmicroinjection and numerous transmembrane carrier techniques.

A “pharmaceutical composition” refers to a mixture of one or more of thecompounds described herein, or physiologically/pharmaceuticallyacceptable salts or prodrugs thereof, with other chemical components,such as physiologically acceptable carriers and excipients. The purposeof a pharmaceutical composition is to facilitate administration of acompound to an organism.

A “prodrug” refers to an agent which is converted into the parent drugin vivo. Prodrugs are often useful because, in some situations, they maybe easier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent drug is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug. An example, without limitation, of a prodrug wouldbe a compound of the present invention which is administered as an ester(the “prodrug”) to facilitate transmittal across a cell membrane wherewater solubility is detrimental to mobility but then is metabolicallyhydrolyzed to the carboxylic acid, the active entity, once inside thecell where water solubility is beneficial.

A further example of a prodrug might be a short polypeptide, forexample, without limitation, a 2-10 amino acid polypeptide, bondedthrough a terminal amino group to a carboxy group of a compound of thisinvention wherein the polypeptide is hydrolyzed or metabolized in vivoto release the active molecule.

As used herein, a “physiologically/pharmaceutically acceptable carrier”refers to a carrier or diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound.

An “excipient” refers to an inert substance added to a pharmaceuticalcomposition to further facilitate administration of a compound.Examples, without limitation, of excipients include calcium carbonate,calcium phosphate, various sugars and types of starch, cellulosederivatives, gelatin, vegetable oils and polyethylene glycols.

“In vitro” refers to procedures performed in an artificial environmentsuch as, e.g., without limitation, in a test tube or culture medium.

As used herein, “in vivo” refers to procedures performed within a livingorganism such as, without limitation, a mouse, rat or rabbit.

As used herein, “PK related disorder,” “PK driven disorder,” and“abnormal PK activity” all refer to a condition characterized byinappropriate, i.e., under or, more commonly, over, PK catalyticactivity, where the particular PK can be an RTK, a CTK or an STK.Inappropriate catalytic activity can arise as the result of either: (1)PK expression in cells which normally do not express PKs, (2) increasedPK expression leading to unwanted cell proliferation, differentiationand/or growth, or, (3) decreased PK expression leading to unwantedreductions in cell proliferation, differentiation and/or growth.Over-activity of a PK refers to either amplification of the geneencoding a particular PK or production of a level of PK activity whichcan correlate with a cell proliferation, differentiation and/or growthdisorder (that is, as the level of the PK increases, the severity of oneor more of the symptoms of the cellular disorder increases).Under-activity is, of course, the converse, wherein the severity of oneor more symptoms of a cellular disorder increase as the level of the PKactivity decreases.

As used herein, the terms “prevent”, “preventing” and “prevention” referto a method for barring an organism from acquiring a PK related disorderin the first place.

As used herein, the terms “treat”, “treating” and “treatment” refer to amethod of alleviating or abrogating a PK mediated cellular disorderand/or its attendant symptoms. With regard particularly to cancer, theseterms simply mean that the life expectancy of an individual affectedwith a cancer will be increased or that one or more of the symptoms ofthe disease will be reduced.

The term “organism” refers to any living entity comprised of at leastone cell. A living organism can be as simple as, for example, a singleeukariotic cell or as complex as a mammal, such as a cat, dog, humanbeing, etc.

The term “therapeutically effective amount” as used herein refers tothat amount of the compound being administered which will relieve tosome extent one or more of the symptoms of the disorder being treated.In reference to the treatment of cancer, a therapeutically effectiveamount refers to that amount which has the effect of (1) reducing thesize of the tumor, (2) inhibiting (that is, slowing to some extent,preferably stopping) tumor metastasis, (3) inhibiting to some extent(that is, slowing to some extent, preferably stopping) tumor growth,and/or, (4) relieving to some extent (or, preferably, eliminating) oneor more symptoms associated with the cancer.

By “monitoring” is meant observing or detecting the effect of contactinga compound with a cell expressing a particular PK. The observed ordetected effect can be a change in cell phenotype, in the catalyticactivity of a PK or a change in the interaction of a PK with a naturalbinding partner. Techniques for observing or detecting such effects arewell-known in the art.

“Cell phenotype” refers to the outward appearance of a cell or tissue orthe biological function of the cell or tissue. Examples, withoutlimitation, of a cell phenotype are cell size, cell growth, cellproliferation, cell differentiation, cell survival, apoptosis, andnutrient uptake and use. Such phenotypic characteristics are measurableby techniques well-known in the art.

A “natural binding partner” refers to a polypeptide that binds to aparticular PK in a cell. Natural binding partners can play a role inpropagating a signal in a PK-mediated signal transduction process. Achange in the interaction of the natural binding partner with the PK canmanifest itself as an increased or decreased concentration of thePK/natural binding partner complex and, as a result, in an observablechange in the ability of the PK to mediate signal transduction.

PREFERRED EMBODIMENTS

At present certain compounds of formula (I) are more preferred. Somesuch preferred embodiments are disclosed below:

(i) A preferred group of compounds is that wherein Het is:

wherein:

A¹ or A² or A³ or A² and A⁴ are nitrogen and the other A's are carbonand the R groups on the A's that are carbon are selected from the groupconsisting of hydrogen, —NH₂ and —C(O)OR²⁶, R²⁶ being selected from thegroup consisting of hydrogen and unsubstituted lower alkyl. Morepreferably Het is 2-, 3-, or 4-pyridyl or 2-, 4-, or 5-pyrimidinyloptionally substituted with an amino or —COOH group. Most preferably4-pyridyl.

(ii) Another presently preferred embodiment of this invention is thatwherein Het is:

wherein:

D is nitrogen or carbon, preferably carbon;

R⁸, R⁹, R¹¹ and R¹² are hydrogen; and

Z is —NR¹⁰ where R¹⁰ is selected from the group consisting of hydrogen,—C(O)R²⁶, —C(O)OR²⁶, —C(O)NR²⁸R²⁹, —C(S)NR²⁸R²⁹, —C(NH)NR²⁸R²⁹ and—S(O)₂R²⁶ where R²⁶, R²⁸, and R²⁹ are as defined in the Summary of theinvention Preferably Het is piperidin-4-yl, piperazin-4-yl, or4-methylpiperazin-1-yl.

(iii) Another presently preferred aspect of this invention is thatwherein Het is:

wherein:

D is carbon, E¹ is sulfur, E⁴ is nitrogen, E² and E³ are carbon, R¹³ andR¹⁶ do not exist and R¹⁴ and R¹⁵ are hydrogen or E² is nitrogen, E⁴ issulfur, E¹ and E³ are carbon, R¹³ is hydrogen, R¹⁴ and R¹⁶ do not existand R¹⁵ is —NR²⁸R²⁹ or E² and E³ are nitrogen, E¹ and E⁴ are carbon, R¹³and R¹⁶ are hydrogen and R¹⁴ and R¹⁵ do not exist. Preferably, Het isthiazol-2-yl.

(iv) Another preferred group of compounds is that wherein Q is:

wherein:

J¹ is nitrogen and J², J³ and J⁴ are carbon.

Within this group a more preferred group of compounds is that whereinR²² is hydrogen.

Within the more preferred group, an even more preferred group ofcompounds is that wherein:

R²³ is selected from the group consisting of hydrogen, unsubstitutedlower alkyl, —C(O)OR²⁶, and —C(O)NR²⁸R²⁹ where R²⁶ is hydrogen orunsubstituted lower alkyl and R²⁸ and R²⁹ are independently selectedfrom the group consisting of hydrogen or unsubstituted lower alkyl or,combined, R²⁸ and R²⁹ form a group selected from the group consisting of—(CH₂)₂N(R³⁰)(CH₂)₂—,

—(CH₂)₂O(CH₂)₂— or —(CH₂)₅—, R³⁰ being selected from the groupconsisting of hydrogen and unsubstituted lower alkyl. Preferably R²³ ishydrogen, methyl, ethyl, carboxy, ethoxycarbonyl, pyridin-1-ylcarbonyl,piperazin-1-ylcarbonyl, or 4-methylpiperazin-1-ylcarbonyl; or

R²³ together with R²⁴ combines to form —(CH₂)₄— and —CH═CH—CR³⁴═CH— R³⁴is selected from the group consisting of hydrogen and —O(CH₂)₂NR²⁸R²⁹and R²⁸ and R²⁹ are independently selected from the group consisting ofhydrogen or unsubstituted lower alkyl or, combined, R²⁸ and R²⁹ form agroup selected from the group consisting of —(CH₂)₂N(R³⁰)(CH₂)₂—,—(CH₂)₂O(CH₂)₂— or —(CH₂)₅—, R³⁰ being selected from the groupconsisting of hydrogen and unsubstituted lower alkyl, preferablyhydrogen or methyl.

(v) Another preferred group of compounds is that wherein R²⁴ and R²⁵ areindependently selected from the group consisting of hydrogen,unsubstituted lower alkyl, aryl optionally substituted with a groupselected from the group consisting of halo, unsubstituted lower alkoxy,morpholino and 4-formylpiperidinyl,

—(CH₂)_(n)C(O)NR²⁸R²⁹, —(CH₂)_(n)C(O)OR²⁶, —(CH₂) NR²⁸R²⁹,—(CH₂)_(n)OR², —C(O)NH(CH₂)_(n)NR²⁸R²⁹,—O(CH₂)_(n)NR²⁸R²⁹—O(CH₂)_(n)OR²⁶ or, combined, a group selected fromthe group consisting of —(CH₂)₂OC(O)—, —(CH₂)₂N(R³⁰)C(O)—, —(CH₂)₅—,—CH═CH—CH═CH— where n is 0 to 3, R²⁶ is selected from the groupconsisting of hydrogen and unsubstituted lower alkyl and R²⁸ and R²⁹ areindependently selected from the group consisting of hydrogen,unsubstituted lower alkyl, lower alkyl substituted with a phenyl orpyridyl group or —NRR where each R is independently hydrogen orunsubstituted lower alkyl; or R²⁸ and R²⁹ combine to form a groupselected from the group consisting of —(CH₂)₅—, —(CH₂)₂NR³⁰(CH₂)₂— and—(CH₂)₂O(CH₂)₂— where R³⁰ is selected from the group consisting ofhydrogen, unsubstituted lower alkyl and —C(O)R²⁶.

Preferably Q is3,5-dimethyl-4-(4-methylpiperazin-1-yl-carbonyl)-1H-pyrrol-2-yl,5-(methyl-3H-imidazol-4-yl)-1H-pyrrol-2-yl,3-methyl-4-(4-methylpiperidin-1-yl-carbonyl)-1H-pyrrol-2-yl,3,5-dimethyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4,5,6,7-tetrahydro-1H-indol-2-yl,3-(2-carboxyethyl)-5-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-5-ethyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4-ethoxycarbonyl-5-methyl-1H-pyrrol-2-yl,4-(2-carboxyethyl)-3,5-dimethyl-1H-pyrrol-2-yl,4-(carboxymethyl)-3,5-dimethyl-1H-pyrrol-2-yl, indol-2-yl,4,5,6,7-tetrahydroindol-2-yl, 5-(2-morpholin-4-ylethyloxy)indol-2-yl,3-(carboxy)-5-methyl-1H-pyrrol-2-yl, 5-carboxy-3-methyl-1H-pyrrol-2-yl,3-(3-morpholin-4-ylpropyl)-4,5,6,7-tetrahydroindol-2-yl,4-(2-diethylaminoethylaminocarbonyl)-3,5-dimethyl-1H-pyrrol-2-yl,4-(4-methylpiperazin-1-ylcarbonyl)-3,5-dimethyl-1H-pyrrol-2-yl,5-(4-methylpiperazin-1-ylcarbonyl)-3-methyl-1H-pyrrol-2-yl,5-(ethoxycarbonyl)-4,5,6,7-tetrahydro-2H-isoindol-3-yl,4-(pyridin-4-ylaminocarbonyl)-3-phenyl-5-methyl-1H-pyrrol-2-yl,5-methylthiophen-2-yl,3-(2-carboxyethyl)-5-ethoxycarbonyl-4-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4-carboxy-1H-pyrrol-2-yl,3-(4-hydroxyphenyl)-4-ethoxycarbonyl-1H-pyrrol-2-yl,4-(morpholin-4-ylcarbonyl)-3-methyl-1H-pyrrol-2-yl,4-(piperidin-1-ylcarbonyl)-3-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-5-(ethoxycarbonyl)-4-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4-(carboxy)-1H-pyrrol-2-yl,3-(methyl)-4-(benzylaminocarbonyl)-1H-pyrrol-2-yl,3-methyl-4-(pyridin-4-ylmethylaminocarbonyl)-1H-pyrrol-2-yl,3-methyl-4-[3-(2-oxopyrrolidin-1-yl)propyl-aminocarbonyl)-1H-pyrrol-2-yl,5-methyl-4-ethoxycarbonyl-3-[3-(4-methylpiperazin-1-yl)propyl]-1H-pyrrol-2-yl,or 3,5-dimethyl-4-(4-methylpiperazin-1-ylaminocarbonyl)-1H-pyrrol-2-yl.

(vi) Yet another preferred group of compounds is that wherein Q isselected from the group consisting of:

In the above groups (i-vi), a more preferred group of compounds is thatwherein R¹ and R² are hydrogen.

(vii) Another preferred group of compounds is represented by the formula(Ia):

wherein:

Het is 2-, 3-, or 4-pyridyl, pyrimidin-5-yl, thiazol-2-yl, or 2-, 3-, or4-piperidinyl; and

Q is either:

(a)

wherein:

J¹ is nitrogen and J², J³ and J⁴ are carbon and other groups are thosedefined in the Summary of the Invention.

Within this group a more preferred group of compounds is that whereinR²² is hydrogen.

Within the more preferred group, an even more preferred group ofcompounds is that wherein:

R²³ is selected from the group consisting of hydrogen, unsubstitutedlower alkyl, —C(O)OR²⁶, and —C(O)NR²⁸R²⁹ where R²⁶ is hydrogen orunsubstituted lower alkyl and R²⁸ and R²⁹ are independently selectedfrom the group consisting of hydrogen or unsubstituted lower alkyl or,combined, R²⁸ and R²⁹ form a group selected from the group consisting of—(CH₂)₂N(R³⁰)(CH₂)₂—,

—(CH₂)₂O(CH₂)₂— or —(CH₂)₅—, R³⁰ being selected from the groupconsisting of hydrogen and unsubstituted lower alkyl. Preferably R²³ ishydrogen, methyl, ethyl, carboxy, ethoxycarbonyl, pyridin-1-ylcarbonyl,piperazin-1-ylcarbonyl, or 4-methylpiperazin-1-ylcarbonyl; or R²³together with R²⁴ combines to form —(CH₂)₄— and —CH═CH—CR³⁴═CH— R³⁴ isselected from the group consisting of hydrogen and —O(CH₂)₂NR²⁸R²⁹ andR²⁸ and R²⁹ are independently selected from the group consisting ofhydrogen or unsubstituted lower alkyl or, combined, R²⁸ and R²⁹ form agroup selected from the group consisting of —(CH₂)₂N(R³⁰)(CH₂)₂—,—(CH₂)₂O(CH₂)₂— or —(CH₂)₅—, R³⁰ being selected from the groupconsisting of hydrogen and unsubstituted lower alkyl, preferablyhydrogen or methyl.

Another even more preferred group of compounds is that wherein R²⁴ andR²⁵ are independently selected from the group consisting of hydrogen,unsubstituted lower alkyl, aryl optionally substituted with a groupselected from the group consisting of halo, unsubstituted lower alkoxy,morpholino and 4-formylpiperidinyl,—(CH₂)_(n)C(O)NR²⁸R²⁹,—(CH₂)_(n)C(O)OR²⁶, —(CH₂)_(n)NR²⁸R²⁹,—(CH₂)_(n)OR²⁶, —C(O)NH(CH₂)_(n)NR²⁸R²⁹, —O(CH₂)_(n)NR²⁸R²⁹,—O(CH₂)_(n)OR²⁶ or, combined, a group selected from the group consistingof —(CH₂)₂OC(O)—, —(CH₂)₂N(R³⁰)C(O)—, —(CH₂)₅—, —CH═CH—CH═CH— where n is0 to 3, R²⁶ is selected from the group consisting of hydrogen andunsubstituted lower alkyl and R²⁸ and R²⁹ are independently selectedfrom the group consisting of hydrogen, unsubstituted lower alkyl, loweralkyl substituted with a phenyl or pyridyl group or —NRR where each R isindependently hydrogen or unsubstituted lower alkyl; or R²⁸ and R²⁹combine to form a group selected from the group consisting of —(CH₂)₅—,—(CH₂)₂NR³⁰ (CH₂)₂— and —(CH₂)₂O(CH₂)₂— where R³⁰ is selected from thegroup consisting of hydrogen, unsubstituted lower alkyl and —C(O)R²⁶.

Preferably Q is3,5-dimethyl-4-(4-methylpiperazin-1-yl-carbonyl)-1H-pyrrol-2-yl,5-(methyl-3H-imidazol-4-yl)-1H-pyrrol-2-yl,3-methyl-4-(4-methylpiperidin-1-yl-carbonyl)-1H-pyrrol-2-yl,3,5-dimethyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4,5,6,7-tetrahydro-1H-indol-2-yl,3-(2-carboxyethyl)-5-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-5-ethyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4-ethoxycarbonyl-5-methyl-1H-pyrrol-2-yl,4-(2-carboxyethyl)-3,5-dimethyl-1H-pyrrol-2-yl,4-(carboxymethyl)-3,5-dimethyl-1H-pyrrol-2-yl, indol-2-yl,4,5,6,7-tetrahydroindol-2-yl, 5-(2-morpholin-4-ylethyloxy)indol-2-yl,3-(carboxy)-5-methyl-1H-pyrrol-2-yl, 5-carboxy-3-methyl-1H-pyrrol-2-yl,3-(3-morpholin-4-ylpropyl)-4,5,6,7-tetrahydroindol-2-yl,4-(2-diethylaminoethylaminocarbonyl)-3,5-dimethyl-1H-pyrrol-2-yl,4-(4-methylpiperazin-1-ylcarbonyl)-3,5-dimethyl-1H-pyrrol-2-yl,5-(4-methylpiperazin-1-ylcarbonyl)-3-methyl-1H-pyrrol-2-yl,5-(ethoxycarbonyl)-4,5,6,7-tetrahydro-2H-isoindol-3-yl,4-(pyridin-4-ylaminocarbonyl)-3-phenyl-5-methyl-1H-pyrrol-2-yl,5-methylthiophen-2-yl,3-(2-carboxyethyl)-5-ethoxycarbonyl-4-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4-carboxy-1H-pyrrol-2-yl,3-(4-hydroxyphenyl)-4-ethoxycarbonyl-1H-pyrrol-2-yl,4-(morpholin-4-ylcarbonyl)-3-methyl-1H-pyrrol-2-yl,4-(piperidin-1-ylcarbonyl)-3-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-5-(ethoxycarbonyl)-4-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4-(carboxy)-1H-pyrrol-2-yl,3-(methyl)-4-(benzylaminocarbonyl)-1H-pyrrol-2-yl,3-methyl-4-(pyridin-4-ylmethylaminocarbonyl)-1H-pyrrol-2-yl,3-methyl-4-[3-(2-oxopyrrolidin-1-yl)propyl-aminocarbonyl)-1H-pyrrol-2-yl,5-methyl-4-ethoxycarbonyl-3-[3-(4-methylpiperazin-1-yl)propyl]-1H-pyrrol-2-yl,or 3,5-dimethyl-4-(4-methylpiperazin-1-ylaminocarbonyl)-1H-pyrrol-2-yl.

Representative compounds of the invention are disclosed in the Tablebelow:

TABLE 1 Compound Structure Name 1

3-(3,5-Dimethyl-4-(4-methyl-piperazine-1-carbo-nyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-4-yl-1,3-di- hydro-indol-2-one 2

3-(5-Methyl-3H-imidazol-4-ylmethylene)-4-py-ridin-4-yl-1,3-dihydro-indol-2-one 3

1-(2-Oxo-4-pyridin-4-yl-1,2-dihydro-indol-3-ylidene-methyl)-6,7-dihydro-2H-pyrano[3,4-c]pyr- rol-4-one 4

3-[3-Methyl-4-(piperidine-1-carbonyl)-1H-pyr-rol-2-ylmethylene]-4-pyridin-4-yl-1,3-di- hydro-indol-2-one 5

3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-4-pyri-din-4-yl-1,3-dihydro-indol-2-one 6

3-[2-(2-Oxo-4-pyridin-4-yl-1,2-dihydro-indol-3ylidene-methyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-pro- pionic acid 7

3-[5-Methyl-2-(2-oxo-4-pyridin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]pro- pionic acid 8

3-[5-Ethyl-2-(2-oxo-4-pyridin-4-yl-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]-pro- pionic acid 9

4-(2-Carboxy-ethyl)-2-methyl-5-(2-oxo-4-py-ridin-4-yl-1,2-dihydro-indol-3-ylidene- methyl)-1H-pyrrole-3-carboxylicacid ethyl ester 10

3-[2,4-Dimethyl-5-(2-oxo-4-pyridin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]pro- pionic acid 11

[2,4-Dimethyl-5-(2-oxo-4-pyridin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]ace- tic acid 12

3-(1H-Indol-2-ylmethylene)-4-pyridin-4-yl-1,3-di- hydro-indol-2-one 13

4-Pyridin-4-yl-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethyl-ene)-1,3-dihydro-indol-2-one 14

3-[5-(2-Morpholin-4-yl-ethoxy)-1H-indol-2-ylmethyl-ene]-4-pyridin-4-yl-1,3-dihydro-indol-2-one 15

4-Methyl-5-(2-oxo-4-pyridin-4-yl-1,2-dihydro-in-dol-3-ylidenemethyl)-1H-pyrrole-2-caroboxylic acid 16

5-Methyl-2-(2-oxo-4-pyridin-4-yl-1,2-dihydro-in-dol-3-ylidenemethyl)-1H-pyrrole-3-carobxylic acid 17

3-[3-(3-Morpholin-4-yl-propyl)-4,5,6,7-tetra-hydro-1H-indol-2-ylmethylene]-4-pyridin-4-yl-1,3-di- hydro-indol-2-one18

2,4-Dimethyl-5-(2-oxo-4-pyridin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-car- boxylic acid(2-diethylamino-ethyl)-amide 19

3-[3,5-Dimethyl-4-(4-methyl-piperazine-1-car-bonyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-4yl-1,3-di- indol-2-one 20

3-[3-Methyl-5-(4-methyl-piperazine-1-carbonyl)1H-pyr-rol-2-ylmethylene]-4-pyridin-4-yl-1,3-di- hydro-indol-2-one 21

3-(2-Oxo-4-pyridin-4-yl-1,2-dihydro-indol-3-ylidene-methyl)-4,5,6,7-tetrahydro-2H-isoindole1-carbox- ylic acid ethyl ester22

2-Methyl-5-(2-oxo-4-pyridin-4-yl-1,2-dihydro-in-dol-3-ylidenemethyl)-4-phenyl-1H-pyrrole-3-car- boxylic acidpyridin-4-ylamide 23

3-(5-Methyl-thiophen-2-ylmethylene)-4-pyridin-4-yl-1,3-di-hydro-indol-2-one 24

3-(4-Morpholin-4-yl-benzylidene)-4-pyridin-4-yl-1,3-di-hydro-indol-2-one 25

4-[4-(2-Oxo-4-pyridin-4-yl-1,2-dihydro-indol-3ylidene-methyl)-phenyl]-piperazine-1-carbaldehyde 26

4-(2-Carboxy-ethyl)-3-methyl-5-(2-oxo-4-pyri-din-4-yl-1,2-dihydro-indol-3-ylidene- methyl)-1H-pyrrole-2-carboxylicacid ethyl ester 27

4-(2-Hydroxy-ethyl)-5-(2-oxo-4-pyridin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-car- boxylic acid 28

4-(4-Methoxy-phenyl)-5-(2-oxo-4-pyridin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-car- boxylic acid ethyl ester29

3-(5-Methyl-3H-imidazol-4-ylmethylene)-4-pipe-ridin-4-yl-1,3-dihydro-indol-2-one 30

3-[3-Methyl-4-(piperidine-1-carbonyl)-1H-pyr-rol-2-ylmethylene]-4-piperidin-4-yl-1,3-di- hydro-indol-2-one 31

3-[3-Methyl-4-(morpholine-4-carbonyl)-1H-pyr-rol-2-ylmethylene]-4-piperidin-4-yl-1,3-dihydro-indol-2-one 32

1-(2-Oxo-4-piperidin-4-yl-1,2-dihydro-indol-3-ylidene-methyl)-6,7-dihydro-2H-pyrano[3,4-c]pyr- rol-4-one 33

1-(2-Oxo-4-piperidin-4-yl-1,2-dihydro-indol-3-ylidene-methyl)-2,5,6,7-tetrahydro-pyrrolo[3,4-c]pyri- din-4-one 34

5-Methyl-1-(2-oxo-4-piperidin-4-yl-1,2-dihydro-indol-3-ylidenemethyl)-2,5,6,7-tetrahydro-pyr- rolo[3,4-c]pyridin-4-one35

3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-4-pipe-ridin-4-yl-1,3-dihydro-indol-2-one 36

3-[2-(2-Oxo-4-piperidin-4-yl-1,2-dihydro-indol-3-ylidene-methyl)-4,5,6,7-tetrahydro-1H-indol-3yl]-pro- pionic acid 37

3-[5-Methyl-2-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]pro- pionic acid 38

3-[5-Ethyl-2-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]pro- pionic acid 39

4-(2-Carboxy-ethyl)-2-methyl-5-(2-oxo-4-pipe-ridin-4-yl-1,2-dihydro-indol-3-ylidene- methyl)-1H-pyrrole-3-carboxylicacid ethyl ester 40

3-[2,4-Dimethyl-5-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]pro- pionic acid 41

[2,4-Dimethyl-5-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl]ace- tic acid 42

3-(1H-Indol-2-ylmethylene)-4-piperidin-4-yl-1,3-di- hydro-indol-2-one 43

4-Piperidin-4-yl-3-(4,5,6,7-tetrahydro-1H-in-dol-2-ylmethyl)-1,3-dihydro-indol-2-one 44

3-[5-(2-Morpholin-4-yl-ethoxy)-1H-indol-2-ylmethy-lene]-4-piperidin-4-yl-1,3-dihydro- indol-2-one 45

4-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylic acid 46

5-Methyl-2-(2-oxo-4-piperidin-4-yl-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid 47

3-[3-(3-Morpholin-4-yl-propyl)-4,5,6,7-tetra-hydro-1H-indol-2-ylmethylene]-4-piperidin 4-yl-1,3-dihydro-indol-2-one48

2,4-Dimethyl-5-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-car- boxylic acid(2-diethylamino-ethyl)-amide 49

3-[3-Methyl-5-(4-methyl-piperazine-1-carbonyl)1H-pyrrol-2-ylmethyl]-4-piperidin-4-yl-1,3-di- hydro-indol-2-one 50

3-(2-Oxo-4-piperidin-4-yl-1,2-dihydro-indol-3-ylidene-methyl)-4,5,6,7-tetrahydro-2H-isoindole 1-carboxylic acid ethyl ester 51

2-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-4-phenyl-1H-pyrrole-3-car- boxylic acidpyridin-4-ylamide 52

3-(5-Methyl-thiophen-2-ylmethylene)-4-pipe-ridin-4-yl-1,3-dihydro-indol-2-one 53

3-(4-Morpholin-4-yl-benzylidene)-4-piperidin-4yl-1,3-di-hydro-indol-2-one 54

4-(2-Carboxy-ethyl)-3-methyl-5-(2-oxo-4-pipe-ridin-4-yl-1,2-dihydro-indol-3-ylidene- methyl)-1H-pyrrole-2-carboxylicacid ethyl ester 55

4-(2-Hydroxy-ethyl)-5-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-car- boxylic acid 56

4-(4-Methoxy-phenyl)-5-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-car- boxylic acid ethyl ester57

4-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid benzylamide 58

4-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid(pyridin-4-ylmethyl)-amide 59

4-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicacid[3-(2-oxo-pyrrolidin-1-yl)-propyl]-amide 60

2-Methyl-4-[3-(4-methyl-piperazin-1-yl)-pro-pyl]-5-(2-oxo-4-pyridin-2-yl-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid ethyl ester 61

3-[3,5-Dimethyl-4-(4-methyl-piperazine-1-car-bonyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-2yl-1,3-di- hydro-indol-2-one62

3-[3,5-Dimethyl-4-(4-methyl-piperazine-1-car-bonyl)-1H-pyrrol-2-ylmethylene]-4-pyri-midin5-yl-1,3-dihydro-indol-2-one 63

3-[3,5-Dimethyl-4-(4-methyl-piperazine-1-car-bonyl)-1H-pyrrol-2-ylmethylene]-4-thiazol-2yl-1,3-di- hydro-indol-2-one64

2-Methyl-4-[3-(4-methyl-piperazin-1-yl)-pro-pyl]-5-(2-oxo-4-pyrimidin-5-yl-1,2-dihydro-in-dol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid ethyl ester 65

2-Methyl-4-[3-(4-methyl-piperazin-1-yl)-pro-pyl]-5-(2-oxo-4-thiazol-2-yl-1,2-dihydro-in-dol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid ethyl ester 66

5-[4-(6-Amino-pyridin-3-yl)-2-oxo-1,2-dihydro-in-dol-3-ylidenemethyl]-2-methyl-4-[3-(4-meth-ylpiperazin-1-yl)-propyl]-1H-pyrrole-3-car- boxylic acid ethyl ester 67

4-(6-Amino-pyridin-3-yl)-3-[3,5-dimethyl-4-(4-meth-yl-piperazine-1-carbonyl)-1H-pyrrol-2-yl-methylene]-1,3-dihydro-indol-2-one 68

2-Methyl-4-[3-(4-methyl-piperazin-1-yl)-pro-pyl]-5-(2-oxo-4-pyridin-3-yl-1,2-dihydro-in-dol-3-ylidenemethyl)-1H-pyrrole-3-carboxylic acid ethyl ester 69

3-[3,5-Dimethyl-4-(4-methyl-piperazine-1-car-bonyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-3yl-1,3-di- hydro-indol-2-one70

5-(3-{4-Ethoxycarbonyl-5-methyl-3-[3-(4-methylpipe-razin-1-yl)-propyl]-1H-pyrrol-2-yl-methylene}-2-oxo-2,3-dihydro-1H-indol-4-yl)-nico- tinic acid 71

5-{3-[3,5-Dimethyl-4-(4-methyl-piperazine-1-car-bonyl)-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-di-hydro-1H-indol-4-yl}-nicotinic acid 72

5-{3-[4-(2-Diethylamino-ethylcarbamoyl)-3,5-di-methyl-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-di-hydro-1H-indol-4-yl}-nicotinic acid 73

5-{4-(2-Amino-pyrimidin-5-yl)-2-oxo-1,2-di-hydro-indol-3-ylidenemethyl]-2,4-dimethyl-1H-pyr- role-3-carboxylicacid(2-diethylamino-eth- yl)-amide 74

4-(2-Amino-pyrimidin-5-yl)-3-[3,5-dimethyl-4-(4-meth-yl-piperazine-1-carbonyl)-1H-pyrrol-2-ylmeth-ylene]-1,3-dihydro-indol-2-one 75

2,4-Dimethyl-5-(2-oxo-4-pyridin-3-yl-1,2-di-hydro-indol-3-ylidenemethyl)-1H-pyrrole-3-car- boxylicacid(2-diethylamino-ethyl)-amide

1. Brief Description of the Tables

TABLE 1 shows the chemical structures of some exemplary compounds ofthis invention. The compound numbers correspond to the Example numbersin the Examples section. That is, the synthesis of Compound 1 in Table 1is described in Example 1. The compounds presented in Table 1 areexemplary only and are not to be construed as limiting the scope of thisinvention in any manner.

TABLE 2 shows the results of biological testing of some exemplarycompounds of this invention. The results are reported in terms of IC₅₀,the micromolar (μM) concentration of the compound being tested whichcauses a 50% change in the activity of the target PK compared to theactivity of the PK in a control to which no test compound has beenadded. Specifically, the results shown indicate the concentration of atest compound needed to cause a 50% reduction of the activity of thetarget PK. Bioassays which have been or may be used to evaluatecompounds are described in detail below.

Utility

The PKs whose catalytic activity is modulated by the compounds of thisinvention include protein tyrosine kinases of which there are two types,receptor tyrosine kinases (RTKs) and cellular tyrosine kinases (CTKs),and serine-threonine kinases (STKs). RTK mediated signal transduction isinitiated by extracellular interaction with a specific growth factor(ligand), followed by receptor dimerization, transient stimulation ofthe intrinsic protein tyrosine kinase activity and phosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules and lead to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse (e.g., cell division, metabolic effects on the extracellularmicroenvironment, etc.). See, Schlessinger and Ullrich, 1992, Neuron9:303-391.

It has been shown that tyrosine phosphorylation sites on growth factorreceptors function as high-affinity binding sites for SH2 (src homology)domains of signaling molecules. Fantl et al., 1992, Cell 69:413-423,Songyang et al., 1994, Mol. Cell. Biol. 14:2777-2785), Songyang et al.,1993, Cell 72:767-778, and Koch et al., 1991, Science 252:668-678.Several intracellular substrate proteins that associate with RTKs havebeen identified. They may be divided into two principal groups: (1)substrates that have a catalytic domain, and (2) substrates which lacksuch domain but which serve as adapters and associate with catalyticallyactive molecules. Songyang et al., 1993, Cell 72:767-778. Thespecificity of the interactions between receptors and SH2 domains oftheir substrates is determined by the amino acid residues immediatelysurrounding the phosphorylated tyrosine residue. Differences in thebinding affinities between SH2 domains and the amino acid sequencessurrounding the phosphotyrosine residues on particular receptors areconsistent with the observed differences in their substratephosphorylation profiles. Songyang et al., 1993, Cell 72:767-778. Theseobservations suggest that the function of each RTK is determined notonly by its pattern of expression and ligand availability but also bythe array of downstream signal transduction pathways that are activatedby a particular receptor. Thus, phosphorylation provides an importantregulatory step which determines the selectivity of signaling pathwaysrecruited by specific growth factor receptors, as well asdifferentiation factor receptors.

STKs, being primarily cytosolic, affect the internal biochemistry of thecell, often as a down-line response to a PTK event. STKs have beenimplicated in the signaling process which initiates DNA synthesis andsubsequent mitosis leading to cell proliferation.

A group of STKs that comprise a particularly attractive therapeutictarget for cell proliferative disorders are the cyclin dependent kinasesor CDKs. CDKs play a prominent role in control of cellularproliferation. That is, the proliferation of all eukaryotic cells occursthrough a continuum of events called the “cell cycle.” While in fact acontinuum, for purposes of discussion, the cell cycle is convenientlybroken down into four phases, G1, S, G2 and M. There is another phase,known as G₀, which is not part of the cell cycle per se but rather is aquiescent state in which a cell resides prior to entering the cell cycleat G1. In G1, cellular activity is heavily dependent on the stimulatinginfluence of external growth factors. It is during G1 that the machinerynecessary for DNA replication is assembled. Between G1 and S is acritical point called the “restriction” point. At the restriction pointa cell must decide whether it is prepared to continue with the cellcycle. If so, the cell commits to entry into S phase at which time it nolonger requires the stimulation of external growth factors. Progressthrough the cell cycle is entirely intracellular from this point. It isin the S phase that DNA is replicated. At the end of S phase and entryinto G2, a cell has 4N DNA content. In G2, a cell begins preparation forM phase and cytokinesis. Progression through the cell cycle is regulatedby CDKs. As the name suggests, in order to perform their functions, theCDKs require association with cyclin regulatory subunits. Presently,about nine CDKs and about 12 families of cyclins with which the CDKs caninteract are known. Two or these, cyclin D/CDK4 and cyclin E/CDK2 areresponsible for controlling entry of a cell into G1 from G₀, passage ofthe cell through the restriction point and commitment to S phase.Progress through S phase is driven by cyclin E/CDK2 and cyclin A/CDK2,the latter of which promotes completion of S phase and entry into G2.Finally, progression through G2, DNA segregation and eventual separationof the parent cell into two daughter cells during M phase and subsequentcytokinesis is controlled by cyclins A and B in conjunction with CDK1.Throughout the cell cycle there are checkpoints at which a cell monitorsboth its external and internal environments to assure that continuedprogress through the cycle is appropriate. Two important, well-studiedcheck points occur in G1 and G2/mitosis. At the G1 checkpoint, the cellchecks to see that it has adequate nutrition, that it is properlyinteracting with other cells or their substratum and that its DNA isintact. At the G2 checkpoint, the cell assures that DNA replication iscomplete and correct and that the mitotic spindle has properly formed. Anegative response at any of these checkpoints results in arrest of thecell cycle which can be temporary, if repairs can be made, or permanent,that is, death of the cell, if repairs cannot be made. These checkpointsare important because inappropriate cell cycle progress is a hallmark ofcell proliferation disorders such as malignant tumor growth. Since CDKsare primarily responsible for driving cells through the cell cycle,including the checkpoints, their proper functioning is critical toproper cell proliferation. It is for this reason that CDKs haveattracted much interest as therapeutic targets. While therapeuticpotential exists in all the CDKs, CDK2 has come under particularscrutiny due to the apparently critical role that it play in the cellcycle. For example, it has been demonstrated that CDK2 dominant negativeconstructs can halt cell cycle progression completely (S. Van denHeuval, et al., Science, 1993, 262:2050-2054). Furthermore,anchorage-independent growth, a key feature of tumor cells, is mediatedby CDK2 complexes (G. Orend, et al., Oncogene, 1998, 16:2575-2583). Inanother study, a peptide inhibitor of CDK2 function was shown toselectively kill tumor cells over normal cells (Y. N. Chen, et al.,Proc. Natl. Acad. Sci. USA, 1999, 96:4221-4223).

While not being bound to any theory, a possible mechanism by which acompound capable of mediating CDK2 function might act can be deducedfrom the relationship of CDK2 and the tumor suppression gene p53. If DNAto be replicated has been damaged or if the cell is being stimulated byan oncogene, p53 is activated by the cell and expresses a protein whicheither suppresses further cell division or simply instructs the cell tokill itself (apoptosis). However, CDK-2 inhibits the activity of p53,thereby keeping it from performing this crucial function and stimulatingcell growth. To counter this, p53 protein stimulates the production ofanother protein, p21, which complexes with CDK2, thereby inactivatingit. However, when the p53 gene is damaged (e.g., mutated, a conditionfound in most tumor types), the p53-p21/CDK2 complexcell/division-inhibition cascade cannot occur and CDK will stimulate thecell, even though damaged, to divide. This can lead to uncontrolledcellular proliferation and cancer. An exogenous CDK2 inhibitor could, inessence, take the place of p53 and prevent the formation of a canceroustumor. Thus, one aspect of this invention is a compound which inhibitsCDK2 function and thereby the formation of malignant tumors.

Thus it can be seen that PK signal transduction results in, among otherresponses, cell proliferation, differentiation, growth and metabolism.Abnormal cell proliferation may result in a wide array of disorders anddiseases, including the development of neoplasia such as carcinoma,sarcoma, glioblastoma and hemangioma, disorders such as leukemia,psoriasis, arteriosclerosis, arthritis and diabetic retinopathy andother disorders related to uncontrolled angiogenesis and/orvasculogenesis.

A precise understanding of the mechanism by which the compounds of thisinvention inhibit PKs is not required in order to practice the presentinvention. However, while not hereby being bound to any particularmechanism or theory, it is believed that the compounds interact with theamino acids in the catalytic region of PKs. PKs typically possess abi-lobate structure wherein ATP appears to bind in the cleft between thetwo lobes in a region where the amino acids are conserved among PKs.Inhibitors of PKs are believed to bind by non-covalent interactions suchas hydrogen bonding, van der Waals forces and ionic interactions in thesame general region where the aforesaid ATP binds to the PKs. Morespecifically, it is thought that the 2-indolinone component of thecompounds of this invention binds in the region normally occupied by theadenine ring of ATP. Specificity of a particular molecule for aparticular PK may then arise as the result of additional interactionsbetween the various substituents on the 2-indolinone core and the aminoacid domains specific to particular PKs. Thus, different indolinonesubstituents may contribute to preferential binding to particular PKs.The ability to select compounds capable of binding to the ATP (or othernucleotide) binding site makes the compounds of this invention usefulfor targeting any protein with such a site. The compounds disclosedherein may thus have utility as in vitro assays for such proteins aswell as exhibiting in vivo therapeutic effects through interaction withsuch proteins.

In another aspect, the protein kinase, the catalytic activity of whichis modulated by contact with a compound of this invention, is a proteintyrosine kinase, more particularly, a receptor protein tyrosine kinase.Among the receptor protein tyrosine kinases whose catalytic activity canbe modulated with a compound of this invention, or salt thereof, are,without limitation, EGF, HER2,HER3,HER4, IR, IGF-1R, IRR, PDGFRα,PDGFRβ, CSFIR, C-Kit, C-fms, Flk-1R, Flk4, KDR/Flk-1, Flt-1, FGFR-1R,FGFR-2R, FGFR-3R and FGFR-4R.

The protein tyrosine kinase whose catalytic activity is modulated bycontact with a compound of this invention, or a salt or a prodrugthereof, can also be a non-receptor or cellular protein tyrosine kinase(CTK). Thus, the catalytic activity of CTKs such as, without limitation,Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn,Lck, Blk, Hck, Fgr and Yrk, may be modulated by contact with a compoundor salt of this invention.

Still another group of PKs which may have their catalytic activitymodulated by contact with a compound of this invention are theserine-threonine protein kinases such as, without limitation, CDK2 andRaf.

This invention is therefore directed to compounds that modulate PKsignal transduction by affecting the enzymatic activity of RTKs, CTKsand/or STKs, thereby interfering with the signals transduced by suchproteins. More particularly, the present invention is directed tocompounds which modulate RTK, CTK and/or STK mediated signaltransduction pathways as a therapeutic approach to the treatment of manykinds of solid tumors, including but not limited to carcinomas, sarcomasincluding Kaposi's sarcoma, erythroblastoma, glioblastoma, meningioma,astrocytoma, melanoma and myoblastoma. Treatment or prevention ofnon-solid tumor cancers such as leukemia are also contemplated by thisinvention. Indications may include, but are not limited to braincancers, bladder cancers, ovarian cancers, gastric cancers, pancreascancers, colon cancers, blood cancers, lung cancers and bone cancers.

Further examples, without limitation, of the types of disorders relatedto inappropriate PK activity that the compounds described herein may beuseful in preventing, treating and studying, are cell proliferativedisorders, fibrotic disorders and metabolic disorders.

Cell proliferative disorders, which may be prevented, treated or furtherstudied by the present invention include cancer, blood vesselproliferative disorders and mesangial cell proliferative disorders.

Blood vessel proliferative disorders refer to disorders related toabnormal vasculogenesis (blood vessel formation) and angiogenesis(spreading of blood vessels). While vasculogenesis and angiogenesis playimportant roles in a variety of normal physiological processes such asembryonic development, corpus luteum formation, wound healing and organregeneration, they also play a pivotal role in cancer development wherethey result in the formation of new capillaries needed to keep a tumoralive. Other examples of blood vessel proliferation disorders includearthritis, where new capillary blood vessels invade the joint anddestroy cartilage, and ocular diseases, like diabetic retinopathy, wherenew capillaries in the retina invade the vitreous, bleed and causeblindness.

Two structurally related RTKs have been identified to bind VEGF withhigh affinity: the fms-like tyrosine 1 (fit-l) receptor (Shibuya et al.,1990, Oncogene, 5:519-524; De Vries et al., 1992, Science, 255:989-991)and the KDR/FLK-1 receptor, also known as VEGF-R^(2.) Vascularendothelial growth factor (VEGF) has been reported to be an endothelialcell specific mitogen with in vitro endothelial cell growth promotingactivity. Ferrara & Henzel, 1989, Biochein. Biophys. Res. Comm.,161:851-858; Vaisman et al., 1990, J. Biol. Chem., 265:19461-19566.Information set forth in U.S. application Ser. Nos. 08/193,829,08/038,596 and 07/975,750, strongly suggest that VEGF is not onlyresponsible for endothelial cell proliferation, but also is the primeregulator of normal and pathological angiogenesis. See generally,Klagsburn & Soker, 1993, Current Biology, 3(10)699-702;Houck, et al.,1992, J. Biol. Chem., 267:26031-26037.

Normal vasculogenesis and angiogenesis play important roles in a varietyof physiological processes such as embryonic development, wound healing,organ regeneration and female reproductive processes such as follicledevelopment in the corpus luteum during ovulation and placental growthafter pregnancy. Folkman & Shing, 1992, J. Biological Chem.,267(16):10931-34. Uncontrolled vasculogenesis and/or angiogenesis hasbeen associated with diseases such as diabetes as well as with malignantsolid tumors that rely on vascularization for growth. Klagsburn & Soker,1993, Current Biology, 3(10):699-702; Folkham, 1991, J. Natl. CancerInst., 82:4-6; Weidner, et al., 1991, New Engl. J. Med., 324:1-5.

The surmised role of VEGF in endothelial cell proliferation andmigration during angiogenesis and vasculogenesis indicates an importantrole for the KDR/FLK-1 receptor in these processes. Diseases such asdiabetes mellitus (Folkman, 198, in XIth Congress of Thrombosis andHaemostasis(Verstraeta, et al., eds.), pp. 583-596, Leuven UniversityPress, Leuven) and arthritis, as well as malignant tumor growth mayresult from uncontrolled angiogenesis. See e.g., Folkman, 1971, N. Engl.J. Med., 285:1182-1186. The receptors to which VEGF specifically bindsare an important and powerful therapeutic target for the regulation andmodulation of vasculogenesis and/or angiogenesis and a variety of severediseases which involve abnormal cellular growth caused by suchprocesses. Plowman, et al., 1994, DN&P, 7(6):334-339. More particularly,the KDR/FLK-1 receptor's highly specific role in neovascularization makeit a choice target for therapeutic approaches to the treatment of cancerand other diseases which involve the uncontrolled formation of bloodvessels.

Thus, one aspect of the present invention relates to compounds capableof regulating and/or modulating tyrosine kinase signal transductionincluding KDR/FLK-1 receptor signal transduction in order to inhibit orpromote angiogenesis and/or vasculogenesis, that is, compounds thatinhibit, prevent, or interfere with the signal transduced by KDR/FLK-1when activated by ligands such as VEGF. Although it is believed that thecompounds of the present invention act on a receptor or other componentalong the tyrosine kinase signal transduction pathway, they may also actdirectly on the tumor cells that result from uncontrolled angiogenesis.

Although the nomenclature of the human and murine counterparts of thegeneric “flk-I” receptor differ, they are, in many respects,interchangeable. The murine receptor, Flk-1, and its human counterpart,KDR, share a sequence homology of 93.4% within the intracellular domain.Likewise, murine FLK-I binds human VEGF with the same affinity as mouseVEGF, and accordingly, is activated by the ligand derived from eitherspecies. Millauer et al., 1993, Cell, 72:835-846; Quinn et al., 1993,Proc. Natl. Acad. Sci. USA, 90:7533-7537. FLK-1 also associates with andsubsequently tyrosine phosphorylates human RTK substrates (e.g., PLC-γor p85) when co-expressed in 293 cells (human embryonal kidneyfibroblasts).

Models which rely upon the FLK-1 receptor therefore are directlyapplicable to understanding the KDR receptor. For example, use of themurine FLK-1 receptor in methods which identify compounds that regulatethe murine signal transduction pathway are directly applicable to theidentification of compounds which may be used to regulate the humansignal transduction pathway, that is, which regulate activity related tothe KDR receptor. Thus, chemical compounds identified as inhibitors ofKDR/FLK-1 in vitro, can be confirmed in suitable in vivo models. Both invivo mouse and rat animal models have been demonstrated to be ofexcellent value for the examination of the clinical potential of agentsacting on the KDR/FLK-1 induced signal transduction pathway.

Thus, in one aspect, this invention is directed to compounds thatregulate, modulate and/or inhibit vasculogenesis and/or angiogenesis byaffecting the enzymatic activity of the KDR/FLK-1 receptor andinterfering with the signal transduced by KDR/FLK-1. In another aspect,the present invention is directed to compounds which regulate, modulateand/or inhibit the KDR/FLK-1 mediated signal transduction pathway as atherapeutic approach to the treatment of many kinds of solid tumorsincluding, but not limited to, glioblastoma, melanoma and Kaposi'ssarcoma, and ovarian, lung, mammary, prostate, pancreatic, colon andepidermoid carcinoma. In addition, data suggest the administration ofcompounds which inhibit the KDR/Flk-1 mediated signal transductionpathway may also be used in the treatment of hemangioma, restenosis anddiabetic retinopathy.

A further aspect of this invention relates to the inhibition ofvasculogenesis and angiogenesis by other receptor-mediated pathways,including the pathway comprising the flt-l receptor.

Receptor tyrosine kinase mediated signal transduction is initiated byextracellular interaction with a specific growth factor (ligand),followed by receptor dimerization, transient stimulation of theintrinsic protein tyrosine kinase activity and autophosphorylation.Binding sites are thereby created for intracellular signal transductionmolecules which leads to the formation of complexes with a spectrum ofcytoplasmic signaling molecules that facilitate the appropriate cellularresponse, e.g., cell division and metabolic effects to the extracellularmicroenvironment. See, Schlessinger and Ullrich, 1992, Neuron, 9:1-20.

The close homology of the intracellular regions of KDR/FLK-1 with thatof the PDGF-β receptor (50.3% homology) and/or the related flt-lreceptor indicates the induction of overlapping signal transductionpathways. For example, for the PDGF-β receptor, members of the srcfamily (Twamley et al., 1993, Proc. Natl. Acad. Sci. USA, 90:7696-7700),phosphatidylinositol-3′-kinase (Hu et al., 1992, Mol. Cell. Biol.,12:981-990), phospholipase cγ (Kashishian & Cooper, 1993, Mol. Cell.Biol., 4:49-51), ras-GTPase-activating protein, (Kashishian et al.,1992, EMBO J., 11:1373-1382), PTP-ID/syp (Kazlauskas et al., 1993, Proc.Natl. Acad. Sci. USA, 10 90:6939-6943), Grb2 (Arvidsson et al., 1994,Mol. Cell. Biol., 14:6715-6726), and the adapter molecules Shc and Nck(Nishimura et al., 1993, Mol. Cell. Biol., 13:6889-6896), have beenshown to bind to regions involving different autophosphorylation sites.See generally, Claesson-Welsh, 1994, Prog. Growth Factor Res., 5:37-54.Thus, it is likely that signal transduction pathways activated byKDR/FLK-1 include the ras pathway (Rozakis et al., 1992, Nature,360:689-692), the PI-3′-kinase, the src-mediated and the plcγ-mediatedpathways. Each of these pathways may play a critical role in theangiogenic and/or vasculogenic effect of KDR/FLK-1 in endothelial cells.Consequently, a still further aspect of this invention relates to theuse of the organic compounds described herein to modulate angiogenesisand vasculogenesis as such processes are controlled by these pathways.

Conversely, disorders related to the shrinkage, contraction or closingof blood vessels, such as restenosis, are also implicated and may betreated or prevented by the methods of this invention.

Fibrotic disorders refer to the abnormal formation of extracellularmatrices. Examples of fibrotic disorders include hepatic cirrhosis andmesangial cell proliferative disorders. Hepatic cirrhosis ischaracterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar. An increased extracellularmatrix resulting in a hepatic scar can also be caused by a viralinfection such as hepatitis. Lipocytes appear to play a major role inhepatic cirrhosis. Other fibrotic disorders implicated includeatherosclerosis.

Mesangial cell proliferative disorders refer to disorders brought aboutby abnormal proliferation of mesangial cells. Mesangial proliferativedisorders include various human renal diseases such asglomerulonephritis, diabetic nephropathy and malignant nephrosclerosisas well as such disorders as thrombotic microangiopathy syndromes,transplant rejection, and glomerulopathies. The RTK PDGFR has beenimplicated in the maintenance of mesangial cell proliferation. Floege etal., 1993, Kidney International 43:47S-54S.

Many cancers are cell proliferative disorders and, as noted previously,PKs have been associated with cell proliferative disorders. Thus, it isnot surprising that PKs such as, for example, members of the RTK familyhave been associated with the development of cancer. Some of thesereceptors, like EGFR (Tuzi et al., 1991, Br. J. Cancer 63:227-233, Torpet al., 1992, APMIS 100:713-719) HER2/neu (Slamon et al., 1989, Science244:707-712) and PDGF-R (Kumabe et al., 1992, Oncogene, 7:627-633) areover-expressed in many tumors and/or persistently activated by autocrineloops. In fact, in the most common and severe cancers these receptorover-expressions (Akbasak and Suner-Akbasak et al., 1992, J. Neurol.Sci., 111:119-133, Dickson et al., 1992, Cancer Treatment Res.61:249-273, Korc et al., 1992, J. Clin. Invest. 90:1352-1360) andautocrine loops (Lee and Donoghue, 1992, J. Cell. Biol., 118:1057-1070,Korc et al., supra, Akbasak and Suner-Akbasak et al., supra) have beendemonstrated. For example, EGFR has been associated with squamous cellcarcinoma, astrocytoma, glioblastoma, head and neck cancer, lung cancerand bladder cancer. HER2 has been associated with breast, ovarian,gastric, lung, pancreas and bladder cancer. PDGFR has been associatedwith glioblastoma and melanoma as well as lung, ovarian and prostatecancer. The RTK c-met has also been associated with malignant tumorformation. For example, c-met has been associated with, among othercancers, colorectal, thyroid, pancreatic, gastric and hepatocellularcarcinomas and lymphomas. Additionally c-met has been linked toleukemia. Over-expression of the c-met gene has also been detected inpatients with Hodgkins disease and Burkitts disease.

IGF-IR, in addition to being implicated in nutritional support and intype-II diabetes, has also been associated with several types ofcancers. For example, IGF-I has been implicated as an autocrine growthstimulator for several tumor types, e.g. human breast cancer carcinomacells (Arteaga et al., 1989, J. Clin. Invest. 84:1418-1423) and smalllung tumor cells (Macauley et al., 1990, Cancer Res., 50:2511-2517). Inaddition, IGF-I, while integrally involved in the normal growth anddifferentiation of the nervous system, also appears to be an autocrinestimulator of human gliomas. Sandberg-Nordqvist et al., 1993, CancerRes. 53:2475-2478. The importance of IGF-IR and its ligands in cellproliferation is further supported by the fact that many cell types inculture (fibroblasts, epithelial cells, smooth muscle cells,T-lymphocytes, myeloid cells, chondrocytes and osteoblasts (the stemcells of the bone marrow)) are stimulated to grow by IGF-I. Goldring andGoldring, 1991, Eukaryotic Gene Expression, 1:301-326. Baserga andCoppola suggest that IGF-IR plays a central role in the mechanism oftransformation and, as such, could be a preferred target for therapeuticinterventions for a broad spectrum of human malignancies. Baserga, 1995,Cancer Res., 55:249-252, Baserga, 1994, Cell 79:927-930, Coppola et al.,1994, Mol. Cell. Biol., 14:4588-4595.

STKs have been implicated in many types of cancer including, notably,breast cancer (Cance, et al., Int. J. Cancer, 54:571-77 (1993)).

The association between abnormal PK activity and disease is notrestricted to cancer. For example, RTKs have been associated withdiseases such as psoriasis, diabetes mellitus, endometriosis,angiogenesis, atheromatous plaque development, Alzheimer's disease,restenosis, von Hippel-Lindau disease, epidermal hyperproliferation,neurodegenerative diseases, age-related macular degeneration andhemangiomas. For example, EGFR has been indicated in corneal and dermalwound healing. Defects in Insulin-R and IGF-1R are indicated in type-IIdiabetes mellitus. A more complete correlation between specific RTKs andtheir therapeutic indications is set forth in Plowman et al., 1994, DN&P7:334-339.

As noted previously, not only RTKs but CTKs including, but not limitedto, src, abl, fps, yes, fyn, lyn, lck, blk, hck, fgr and yrk (reviewedby Bolen et al., 1992, FASEB J., 6:3403-3409) are involved in theproliferative and metabolic signal transduction pathway and thus couldbe expected, and have been shown, to be involved in many PTK-mediateddisorders to which the present invention is directed. For example,mutated src (v-src) has been shown to be an oncoprotein (pp60^(v-src))in chicken. Moreover, its cellular homolog, the proto-oncogenepp60^(c-src) transmits oncogenic signals of many receptors.Over-expression of EGFR or HER2/neu in tumors leads to the constitutiveactivation of pp60^(c□src), which is characteristic of malignant cellsbut absent in normal cells. On the other hand, mice deficient in theexpression of c-src exhibit an osteopetrotic phenotype, indicating a keyparticipation of c-src in osteoclast function and a possible involvementin related disorders.

Similarly, Zap70 has been implicated in T-cell signaling which mayrelate to autoimmune disorders.

STKs have been associated with inflammation, autoimmune disease,immunoresponses, and hyperproliferation disorders such as restenosis,fibrosis, psoriasis, osteoarthritis and rheumatoid arthritis.

PKs have also been implicated in embryo implantation. Thus, thecompounds of this invention may provide an effective method ofpreventing such embryo implantation and thereby be useful as birthcontrol agents.

Finally, both RTKs and CTKs are currently suspected as being involved inhyperimmune disorders.

A method for identifying a chemical compound that modulates thecatalytic activity of one or more of the above discussed protein kinasesis another aspect of this invention. The method involves contactingcells expressing the desired protein kinase with a compound of thisinvention (or its salt or prodrug) and monitoring the cells for anyeffect that the compound has on them. The effect may be any observable,either to the naked eye or through the use of instrumentation, change orabsence of change in a cell phenotype. The change or absence of changein the cell phenotype monitored may be, for example, without limitation,a change or absence of change in the catalytic activity of the proteinkinase in the cells or a change or absence of change in the interactionof the protein kinase with a natural binding partner.

Examples of the effect of a number of exemplary compounds of thisinvention on several PKs are shown in Table 2. The compounds and datapresented are not to be construed as limiting the scope of thisinvention in any manner whatsoever.

Pharmaceutical Compositions and Use

A compound of the present invention, a prodrug thereof or aphysiologically acceptable salt of either the compound or its prodrug,can be administered as such to a human patient or can be administered inpharmaceutical compositions in which the foregoing materials are mixedwith suitable carriers or excipient(s). Techniques for formulation andadministration of drugs may be found in Remington's PharmacologicalSciences, Mack Publishing Co., Easton, Pa., latest edition.

Routes of Administration:

As used herein, “administer” or “administration” refers to the deliveryof a compound, salt or prodrug of the present invention or of apharmaceutical composition containing a compound, salt or prodrug ofthis invention to an organism for the purpose of prevention or treatmentof a PK-related disorder.

Suitable routes of administration may include, without limitation, oral,rectal, transmucosal or intestinal administration or intramuscular,subcutaneous, intramedullary, intrathecal, direct intraventricular,intravenous, intravitreal, intraperitoneal, intranasal, or intraocularinjections. The preferred routes of administration are oral andparenteral.

Alternatively, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto a solid tumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

Composition/Formulation:

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the compounds of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated by combiningthe active compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, lozenges, dragees, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient. Pharmaceutical preparations for oral use can be made using asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding other suitableauxiliaries if desired, to obtain tablets or dragee cores. Usefulexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol, cellulose preparations such as,for example, maize starch, wheat starch, rice starch and potato starchand other materials such as gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginicacid. A salt such as sodium alginate may also be used.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with a fillersuch as lactose, a binder such as starch, and/or a lubricant such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. Stabilizers may be added in these formulations, also.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray using a pressurized pack or a nebulizer and a suitable propellant,e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetra-fluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds may also be formulated for parenteral administration,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulating materials such assuspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of a water soluble form, such as, without limitation,a salt, of the active compound. Additionally, suspensions of the activecompounds may be prepared in a lipophilic vehicle. Suitable lipophilicvehicles include fatty oils such as sesame oil, synthetic fatty acidesters such as ethyl oleate and triglycerides, or materials such asliposomes. Aqueous injection suspensions may contain substances whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers and/or agents that increase the solubilityof the compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, using, e.g., conventional suppositorybases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as depot preparations. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. A compound of thisinvention may be formulated for this route of administration withsuitable polymeric or hydrophobic materials (for instance, in anemulsion with a pharmacologically acceptable oil), with ion exchangeresins, or as a sparingly soluble derivative such as, withoutlimitation, a sparingly soluble salt.

A non-limiting example of a pharmaceutical carrier for the hydrophobiccompounds of the invention is a cosolvent system comprising benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer and anaqueous phase such as the VPD co-solvent system. VPD is a solution of 3%w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:D5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well, and itself produces low toxicity uponsystemic administration. Naturally, the proportions of such a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of Polysorbate 80, the fraction size ofpolyethylene glycol may be varied, other biocompatible polymers mayreplace polyethylene glycol, e.g., polyvinyl pyrrolidone, and othersugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Inaddition, certain organic solvents such as dimethylsulfoxide also may beemployed, although often at the cost of greater toxicity.

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions herein also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include, but are not limited to, calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols.

Many of the PK modulating compounds of the invention may be provided asphysiologically acceptable salts wherein the claimed compound may formthe negatively or the positively charged species. Examples of salts inwhich the compound forms the positively charged moiety include, withoutlimitation, quaternary ammonium (defined elsewhere herein), salts suchas the hydrochloride, sulfate, carbonate, lactate, tartrate, maleate,succinate wherein the nitrogen atom of the quaternary ammonium group isa nitrogen of the selected compound of this invention which has reactedwith the appropriate acid. Salts in which a compound of this inventionforms the negatively charged species include, without limitation, thesodium, potassium, calcium and magnesium salts formed by the reaction ofa carboxylic acid group in the compound with an appropriate base (e.g.sodium hydroxide (NaOH), potassium hydroxide (KOH), Calcium hydroxide(Ca(OH)₂), etc.).

Dosage:

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in anamount sufficient to achieve the intended purpose, e.g., the modulationof PK activity or the treatment or prevention of a PK-related disorder.

More specifically, a therapeutically effective amount means an amount ofcompound effective to prevent, alleviate or ameliorate symptoms ofdisease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromcell culture assays. Then, the dosage can be formulated for use inanimal models so as to achieve a circulating concentration range thatincludes the IC₅₀ as determined in cell culture (i.e., the concentrationof the test compound which achieves a half-maximal inhibition of the PKactivity). Such information can then be used to more accuratelydetermine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (bothof which are discussed elsewhere herein) for a subject compound. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administrationand dosage can be chosen by the individual physician in view of thepatient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active species which are sufficient to maintain thekinase modulating effects. These plasma levels are referred to asminimal effective concentrations (MECs). The MEC will vary for eachcompound but can be estimated from in vitro data, e.g., theconcentration necessary to achieve 50-90% inhibition of a kinase may beascertained using the assays described herein. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using MEC value. Compoundsshould be administered using a regimen that maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration and other procedures known in the art may be employed todetermine the correct dosage amount and interval.

The amount of a composition administered will, of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration, the judgment of the prescribing physician, etc.

Packaging:

The compositions may, if desired, be presented in a pack or dispenserdevice, such as an FDA approved kit, which may contain one or more unitdosage forms containing the active ingredient. The pack may for examplecomprise metal or plastic foil, such as a blister pack. The pack ordispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accompanied by a notice associatedwith the container in a form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals, which noticeis reflective of approval by the agency of the form of the compositionsor of human or veterinary administration. Such notice, for example, maybe of the labeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a compound of the invention formulated in a compatiblepharmaceutical carrier may also be prepared, placed in an appropriatecontainer, and labeled for treatment of an indicated condition. Suitableconditions indicated on the label may include treatment of a tumor,inhibition of angiogenesis, treatment of fibrosis, diabetes, and thelike.

It is also an aspect of this invention that a compound described herein,or its salt or prodrug, might be combined with other chemotherapeuticagents or cyclooxygenase-2 (COX-2) inhibitors for the treatment of thediseases and disorders discussed above. For instance, a compound, saltor prodrug of this invention might be combined with alkylating agentssuch as fluorouracil (5-FU) alone or in further combination withleukovorin; or other alkylating agents such as, without limitation,other pyrimidine analogs such as UFT, capecitabine, gemcitabine andcytarabine, the alkyl sulfonates, e.g., busulfan (used in the treatmentof chronic granulocytic leukemia), improsulfan and piposulfan;aziridines, e.g., benzodepa, carboquone, meturedepa and uredepa;ethyleneimines and methylmelamines, e.g., altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolmelamine; and the nitrogenmustards, e.g., chlorambucil (used in the treatment of chroniclymphocytic leukemia, primary macroglobulinemia and non-Hodgkin'slymphoma), cyclophosphamide (used in the treatment of Hodgkin's disease,multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lungcancer, Wilm's tumor and rhabdomyosarcoma), estramustine, ifosfamide,novembrichin, prednimustine and uracil mustard (used in the treatment ofprimary thrombocytosis, non-Hodgkin's lymphoma, Hodgkin's disease andovarian cancer); and triazines, e.g., dacarbazine (used in the treatmentof soft tissue sarcoma).

Likewise a compound, salt or prodrug of this invention might be expectedto have a beneficial effect in combination with other antimetabolitechemotherapeutic agents such as, without limitation, folic acid analogs,e.g. methotrexate (used in the treatment of acute lymphocytic leukemia,choriocarcinoma, mycosis fungiodes breast cancer, head and neck cancerand osteogenic sarcoma) and pteropterin; and the purine analogs such asmercaptopurine and thioguanine which find use in the treatment of acutegranulocytic, acute lymphocytic and chronic granulocytic leukemias.

A compound, salt or prodrug of this invention might also be expected toprove efficacious in combination with natural product basedchemotherapeutic agents such as, without limitation, the vincaalkaloids, e.g., vinblastin (used in the treatment of breast andtesticular cancer), vincristine and vindesine; the epipodophylotoxins,e.g., etoposide and teniposide, both of which are useful in thetreatment of testicular cancer and Kaposi's sarcoma; the antibioticchemotherapeutic agents, e.g., daunorubicin, doxorubicin, epirubicin,mitomycin (used to treat stomach, cervix, colon, breast, bladder andpancreatic cancer), dactinomycin, temozolomide, plicamycin, bleomycin(used in the treatment of skin, esophagus and genitourinary tractcancer); and the enzymatic chemotherapeutic agents such asL-asparaginase.

In addition to the above, a compound, salt or prodrug of this inventionmight be expected to have a beneficial effect used in combination withthe platinum coordination complexes (cisplatin, etc.); substituted ureassuch as hydroxyurea; methylhydrazine derivatives, e.g., procarbazine;adrenocortical suppressants, e.g., mitotane, aminoglutethimide; andhormone and hormone antagonists such as the adrenocorticosteriods (e.g.,prednisone), progestins (e.g., hydroxyprogesterone caproate); estrogens(e.g., diethylstilbesterol); antiestrogens such as tamoxifen; androgens,e.g., testosterone propionate; and aromatase inhibitors such asanastrozole.

The combination of a compound of this invention might be expected to beparticularly effective in combination with mitoxantrone or paclitaxelfor the treatment of solid tumor cancers or leukemias such as, withoutlimitation, acute myelogenous (non-lymphocytic) leukemia.

Lastly, in addition to the above, a compound, salt or prodrug of thisinvention might be expected to have a beneficial effect used incombination with COX-2 inhibitor. To treat inflammation. COX-2inhibitors for use in combination with a compound, salt or prodrug ofthe preferred embodiments of the present invention might include,without limitation, those disclosed in WO 96/41626 and U.S. Pat. No.6,248,745. Other COX-2 inhibitors for use in the combinations of theinvention include those disclosed in Drugs of the Future, 1997, 22,711-714 which document is incorporated herein by reference, namelyMeloxicam, L-745337 (Merck), MK-966 (Merck), L-768277 (Merck), GR-253035(Glaxo-Wellcome), JTE-522 (Japan Tobacco), RS-57067-000 (Roche),SC-58125 (Searle), SC-078 (Searle), PD-138387 (Warner-Lambert), NS-398(Taisho), flosulide and PD-164387 (Warner-Lambert).

EXAMPLES

The compounds of this invention may be readily synthesized usingtechniques well known in the chemical arts. It will be appreciated bythose skilled in the art that other synthetic pathways for forming thecompounds of the invention, and others like them, are available and thatthe following is offered by way of example and not limitation. Inaddition, such other synthetic pathways are within the scope of thisinvention.

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and to practice thepresent invention. They should not be considered as limiting the scopeof the invention, but merely as being illustrative and representativethereof.

Synthetic Examples Preparation of 4-heteroarylindolinones

4-Pyridin-4-yl-1,3-dihydroindol-2-one

Palladium catalyst PdCl₂(dppf).CH₂Cl₂ (1.22 g, 1.5 mmol) was added to amixture of 4-bromoindole (9.80 g, 50 mmol), bis(pinacolato)diboron(13.97 g, 55 mmol), and potassium acetate (14.72 g, 150 mmol) in DMSO(200 mL). The system was degassed, and then purged three times withnitrogen. The mixture was stirred at 80° C. in an oil bath undernitrogen for 22 hours. It was then cooled to room temperature and pouredinto water (1 L). The aqueous mixture was extracted with three portionsof ethyl acetate. The combined extracts were washed five times withbrine to remove DMSO and then dried over anhydrous Na₂SO₄. The residuewas purified on a silica gel column, eluting with EtOAc-hexane (9:1), togive 8.01 g (66%) of4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole.

¹H NMR (300 MHz, DMSO-d₆): δ 11.03 (br s, 1H, NH)), 7.49 (d, J=7.7 Hz,1H), 7.38 (dd, J=0.9 & 7.0 Hz, 1H), 7.38 (t, J=2.6 Hz, 1H), 7.06 (dd,J=7.7 & 7.0 Hz, 1H), 6.73 (br d, J=2.2 Hz, 1H), 1.32 (s, 12H, 4CH₃).

MS m/e 244 [M⁺+1].

Palladium catalyst Pd(PPh₃)₄ (1.00 g, 0.87 mmol) and freshly preparedaqueous sodium hydroxide(4.63 g, 115.9 mmol in 42 mL water) were addedto a mixture of4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole (7.03 g, 28.9mmol) and 4-bromopyridine hydrochloride (5.68 g, 29.2 mmol) in THF (98mL). The system was degassed and then purged three times with nitrogen.The mixture was stirred under nitrogen at 70° C. in an oil bath for 6hours. It was then cooled to room temperature and ethyl acetate (400 mL)added. The organic layer was isolated, washed with brine, dried overanhydrous Na₂SO₄ and concentrated. The residue was triturated withdichloromethane to give 5.1 g (91%) of 4-pyridin-4-yl-1H-indole.

¹H NMR (300 MHz, DMSO-d₆) δ 11.39 (br s, 1H, NH), 8.65 (dd, J=1.5 & 4.6Hz, 2H), 7.67 (dd, J=1.6 & 4.6 Hz, 2H), 7.49 (m, 2H), 7.20 (m, 2H), 6.62(br d, J=3.0 Hz, 1H).

Pyridinium tribromide (90% (Aldrich), 24.70 g, 69.50 mmol) was addedportion-wise over 10 minutes to a suspension of 4-pyridin-4-yl-1H-indole(4.50 g, 23.17 mmol) in 2-methyl-2-propanol (135 mL), ethanol (90 mL)and acetic acid (45 mL). The mixture was stirred at room temperature forone hour and then acetic acid (180 mL) was added. After stirring for anadditional hour, water (1 mL) and zinc dust (15.06 g, 232 mmol) wereadded and stirring was continued for another hour. Residual zinc dustwas removed by filtration and washed with methanol. The filtrate wasconcentrated and the syrupy residue was stirred in water (500 mL)overnight. The solid that formed was filtered, washed with water toremove zinc and pyridine salts and dried under high vacuum dry to give5.85 g (99%) of 4-pyridin-4-yl-1,3-dihydroindol-2-one as a light yellowsolid.

¹H NMR (300 MHz, DMSO-d₆) δ 10.54 (br s, 1H, N-H), 8.62 (d, J=5.3 Hz,2H), 7.59 (d, J=5.3 Hz, 2H), 7.32 (dd, J=8.3 & 7.4 Hz, 1H, H-6), 7.10(d, J=8.3 Hz, H-7), 6.89 (d, J=7.4 Hz, H-5), 3.68 (s, 2H, CH₂).

MS m/e 211 [M⁺+1].

4-Piperidin-4-yl-1,3-dihydroindol-2-one

To a suspension of 4-pyridin-4-yl-1,3-dihydroindol-2-one acetic acidsalt (5.50 g, 20.4 mmol) in methanol (160 mL), water (70 mL) and aceticacid (30 mL) was added concentrated hydrochloric acid (2 mL) followed byplatinum(IV) oxide (360 mg). The mixture was hydrogenated for threedays. It was then filtered through celite, which was washed withmethanol. The filtrate was evaporated and dried under high vacuum. Theresidue was dissolved in methanol (500 mL) and treated with a basicresin (hydroxide form) at pH=9-10. The resin was removed by filtrationand washed with methanol. The filtrate was evaporated and dried underhigh vacuum to give 4.2 g (96%) of4-piperidin-4-yl-1,3-dihydroindol-2-one.

MS m/e 217 [M⁺+1].

4-(1-Methylpiperidin-4-yl)-1,3-dihydroindol-2-one

Methyl iodide (130.6 mg, 0.92 mmol) was added to a solution of4-piperidin-4-yl-1,3-dihydroindol-2-one (199 mg, 0.92 mmol) inacetonitrile (10 mL) and methanol (1 mL). The reaction was stirred atroom temperature under nitrogen for 2 days. The solvents were removedand the residue was purified on a silica gel column to give 150 mg (70%)of 4-(1-methylpiperidin-4-yl)-1,3-dihydroindol-2-one as a tan solid.

MS m/z 231 [M⁺+1].

4-Thiazol-2-yl-1,3-dihydroindol-2-one

Palladium catalyst Pd(PPh₃)₄ (142 mg, 0.12 mmol) and freshly preparedaqueous sodium hydroxide (493 mg, 12.33 mmol in 6 mL of water) wereadded to a mixture of4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole (1 g, 4.11mmol) and 2-bromothiazole (809 mg, 4.93 mmol) in THF (20 mL). The systemwas degassed and then purged three times with nitrogen. The mixture wasstirred under nitrogen at 70° C. in an oil bath for 4 hours. It was thencooled to room temperature and ethyl acetate (300 mL) was added. Theorganic layer was isolated, washed with brine, dried over anhydrousNa₂SO₄ and concentrated. The residue was triturated with dichloromethaneto give 350 mg (42%) of 4-thiazol-2-yl-1H-indole as a pale yellow solid.

¹H NMR (400 MHz, DMSO-d₆) δ 11.45 (br s, 1H, NH), 7.99 (d, J=3.3 Hz,1H), 7.79 (d, J=3.3 Hz, 1H), 7.69 (dd, J=1 & 7.2 Hz, 1H), 7.53 (m, 2H),7.21 (t, J=7.7 Hz, 1H), 7.13 (m, 1H).

MS +ve APCI 201 [M⁺+1].

Pyridinium tribromide (90% (Aldrich), 1.9 g, 5.34 mmol) was addedportion-wise over 10 minutes to a suspension of 4-thiazol-2-yl-1H-indole(360 mg, 1.78 mmol) in 2-methyl-2-propanol (15 mL), ethanol (9 mL) andacetic acid (5 mL). The mixture was stirred at room temperature for 2hours after which acetic acid (18 mL), water (1 mL) and zinc dust (1.5g, 23.14 mmol) were added. Stirring was continued for 1.5 hours.Residual zinc dust was removed by filtration and washed with methanol.The filtrate was concentrated and the syrupy residue was stirred inwater (50 mL) overnight. The solid which formed was filtered, washedwith water to remove the zinc and pyridine salts and dried under highvacuum to give 4-thiazol-2-yl-1,3-dihydroindol-2-one.

¹H NMR (400 MHz, DMSO-d₆) δ 10.58 (br s, 1H, NH), 7.99 (d, J=3.2 Hz,1H), 7.86 (d, J=3.2 Hz, 1H), 7.56 (dd, J=0.9 & 7.8 Hz, 1H), 7.32 (t,J=7.8 Hz, 1H), 6.92 (d, J=7.8 Hz, 1H), 3.75 (s, 2H, CH₂).

MS +ve APCI m/z 217 [M⁺+1].

4-Pyrimidin-5-yl-1,3-dihydroindol-2-one

Palladium catalyst Pd(PPh₃)₄ (142 mg, 0.12 mmol) and freshly preparedaqueous sodium hydroxide (493 mg, 12.33 mmol, in 6 mL of water) wereadded to a mixture of4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-1H-indole (1 g, 4.11mmol) and 5-bromopyrimidine (784 mg, 4.93 mmol) in THF (20 mL). Thesystem was degassed and then purged three times with nitrogen. Themixture was stirred under nitrogen at 70° C. in an oil bath for 4 hours.It was then cooled to room temperature and ethyl acetate (300 mL) wasadded. The organic layer was isolated, washed by brine, dried overanhydrous Na₂SO₄ and concentrated. The residue was triturated withdichloromethane to give 570 mg (71%) of 4-pyrimidin-5-yl-1H-indole as apale green solid.

¹H NMR (400 MHz, DMSO-d₆) δ 11.44 (br s, 1H, NH), 9.21 (s, 1H), 9.11 (s,2H), 7.52 (m, 2H), 7.24 (m, 2H), 6.60 (m, 1H).

MS m/z 196 [M⁺+1].

Pyridinium tribromide (90% (Aldrich), 4 g, 11.22 mmol) was addedportion-wise over 10 minutes to a suspension of4-pyrimidin-5-yl-1H-indole (730 mg, 3.74 mmol) in 2-methyl-2-propanol(25 mL), ethanol (15 mL) and acetic acid (9 mL). The mixture was stirredat room temperature for 2 hours after which acetic acid (36 mL), water(1 mL) and zinc (3.2 g, 56.1 mmol) were added. Stirring was continuedfor 1.5 hours. Residual zinc dust was filtered and washed with methanol.The filtrate was concentrated and the syrupy residue was stirred inwater (100 mL) overnight. The precipitate which formed was filtered,washed with water to remove zinc and pyridine salts and dried under highvacuum to give 595 mg (75%) of 4-pyrimidin-5-yl-1,3-dihydroindol-2-oneas a light brown solid.

¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (d, J 3.8 Hz, 2H), 7.84 (m, 1H), 7.44(m, 2H), 3.35 (s, 2H, CH₂).

MS −ve APCI m/z 210 [M⁺−1].

4-(6-Aminopyridin-3-yl)-1,3-dihydroindol-2-one

Palladium catalyst Pd(PPh₃)₄ (142 mg, 0.12 mmol) and freshly preparedaqueous sodium hydroxide (493 mg, 12.33 mmol in 6 mL of water) wereadded to a mixture of4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole (1 g, 4.11mmol) and 2-amino-5-bromopyridine (853 mg, 4.93 mmol) in THF (20 mL).The system was degassed and then purged three times with nitrogen. Themixture was stirred under nitrogen at 80° C. in an oil bath for 4.5hours. It was then cooled to room temperature and ethyl acetate (300 mL)was added. The organic layer was isolated, washed with brine, dried overanhydrous Na₂SO₄ and concentrated. The residue was purified by columnchromatography to give 720 mg (84%) of5-(1H-indol-4-yl)-pyridin-2-ylamine as a thick brown syrup.

¹H NMR (400 MHz, DMSO-d₆) δ 11.18 (br s, 1H, NH), 8.20 (d, J=0.9 Hz,1H), 7.67 (dd, J=2.4 & 8.5 Hz, 1H), 7.36 (t, J=2.8 Hz, 1H), 7.32 (d,J=7.3 Hz, 1H), 7.11 (t, J=7.7 Hz, 1H), 6.97 (d, J=6.5 Hz, 1H), 6.56 (dd,J=0.6 & 7.9 Hz, 1H), 6.50 (m, 1H), 5.99 (s, 2H, NH₂).

MS −APCI m/z 208 [M⁺−1].

Pyridinium tribromide (90% (Aldrich), 3.7 g, 10.32 mmol) was addedportion-wise over 10 minutes to a suspension of5-(1H-indol-4-yl)-pyridin-2-ylamine (720 mg, 3.44 mmol) in2-methyl-2-propanol (25 mL), ethanol (15 mL) and acetic acid (9 mL). Themixture was stirred at room temperature for 2 hours and then acetic acid(36 mL), water (1 mL) and zinc dust (3.2 g, 56.1 mmol) were added.Stirring was continued for 1.5 hours. Residual zinc dust was filteredand washed with methanol. The filtrate was concentrated and the syrupyresidue was stirred in water (100 mL) overnight. The solid that formedwas filtered, washed with water to remove zinc and pyridine salts anddried under high vacuum to give 750 mg (97%) of4-(6-amino-pyridin-3-yl)-1,3-dihydroindol-2-one as a light brown solid.

MS +ve APCI m/z 226 [M⁺+1].

4-Pyridin-2-yl-1,3-dihydroindol-2-one

Palladium catalyst Pd(PPh₃)₄ (714 mg, 0.6 mmol) and freshly preparedaqueous sodium hydroxide (2.47 g, 61.8 mmol in 22 mL of water) wereadded to a mixture of4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole (5 g, 20.6mmol) and 2-bromopyridine (2 mL, 20.8 mmol) in THF (70 mL). The systemwas degassed and then purged three times with nitrogen. The mixture wasstirred under nitrogen at 70° C. in an oil bath for 6 hours. It was thencooled to room temperature and 400 ml ethyl acetate was added. Theorganic layer was isolated, washed with brine, dried over anhydrousNa₂SO₄ and concentrated. The residue was triturated with dichloromethaneto give 2.85 g (71%) of 4-pyridin-2-yl-1H-indole as a pale yellow solid.

¹H NMR (360 MHz, DMSO-d₆) δ 11.27 (br s, 1H, NH), 8.72 (d, J=4.6 Hz,1H), 7.89 (m, 2H), 7.52 (d, J=7.7 Hz, 1H), 7.49 (d, J=7.7 Hz, 1H), 7.44(t, J=2.8 Hz, 1H), 7.33 (m, 1H), 7.21 (t, J=7.7 Hz, 1H), 6.93 (m, 1H).

MS +APCI m/z 195 [M⁺+1].

Pyridinium tribromide (90% (Aldrich), 15.4 g, 43.2 mmol) was addedportion-wise over 10 minutes to a suspension of 4-pyridin-2-yl-1H-indole(2.8 g, 14.4 mmol) in 2-methyl-2-propanol (84 mL), ethanol (56 mL) andacetic acid (28 mL). The mixture was stirred at room temperature for 2hours after which acetic acid (100 mL) was added. Stirring was continuedfor one hour and then water (0.5 mL) and zinc dust (9.4 g, 144 mmol)were added. Stirring was continued for another hour. Residual zinc dustwas filtered and washed with methanol. The filtrate was concentrated andthe syrupy residue was stirred in water(300 mL) overnight. The solidthat formed was filtered, washed with water to remove zinc and pyridinesalts and dried under high vacuum to give 2.8 g (92%) of4-pyridin-2-yl-1,3-dihydroindol-2-one was obtained.

¹H NMR (400 MHz, DMSO-d₆) δ 10.49 (s, 1H, NH), 8.68 (d, J 4.2 Hz, 1H),7.83-7.91 (m, 2H), 7.45 (d, J=7.3 Hz, 1H), 7.29-7.38 (m, 2H), 6.89 (d,J=7.4 Hz, 1H), 3.77 (s, 2H, CH₂).

MS +ve APCI m/z 211 [M⁺+1].

4-Pyridin-3-yl-1,3-dihydroindol-2-one

Palladium catalyst Pd(PPh₃)₄ (284 mg, 0.25 mmol) and freshly preparedaqueous sodium hydroxide (984 mg in 9 mL of water) were added to amixture of 4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indole (2g, 8.2 mmol) and 3-bromopyridine (0.8 mL, 8.3 mmol) in THF (28 mL). Thesystem was degassed and then purged three times with nitrogen. Themixture was stirred under nitrogen at 70° C. in an oil bath for 6 hours.It was then cooled to room temperature and 400 ml ethyl acetate wasadded. The organic layer was isolated, washed with brine, dried overanhydrous Na₂SO₄ and concentrated. The residue was columnchromatographed to give 1 g (62%) of 4-pyridin-3-yl-1H-indole as a whitesolid.

¹H NMR (360 MHz, DMSO-d₆) δ 11.32 (br s, 1H, NH), 8.87 (d, J=2.1 Hz,1H), 8.58 (dd, J=2.1 & 4.8 Hz, 1H), 8.06 (dt, J=2.1 & 8.1 Hz, 1H), 7.52(dd, 1H), 7.46 (m, 2H), 7.22 (t, J=7.6 Hz, 1H), 7.14 (t, J=7.1 Hz, 1H),6.54 (d, J=3.2 Hz, 1H).

MS +ve APCI m/z 195 [M⁺+1].

Pyridinium tribromide (90% (Aldrich), 5.5 g, 15.3 mmol) was addedportion-wise over 10 minutes to a suspension of 4-pyridin-3-yl-1H-indole(1 g, 5.1 mmol) in 2-methyl-2-propanol (30 mL), ethanol (20 mL) andacetic acid (10 mL). The mixture was stirred at room temperature for 2hours and then acetic acid (50 mL) was added. After stirring for anadditional hour, water (0.5 mL) and zinc dust (3.3 g, 51 mmol) wereadded and stirring was continued for another hour. Residual zinc dustwas filtered and washed with methanol. The filtrate was concentrated andthe syrupy residue was stirred in water (100 mL) overnight. The solidwhich formed was filtered, washed with water to remove zinc and pyridinesalts and dried under high vacuum to give 1.1 g (100%) of4-pyridin-3-yl-1,3-dihydro-indol-2-one.

¹H NMR (400 MHz, DMSO-d₆) δ 10.52 (br s, 1H, NH), 8.78 (d, J=1.8 Hz,1H), 8.58 (dd, J=1.5 & 4.7 Hz, 1H), 8.02 (m, 1H), 7.5 (m, 1H), 7.31 (t,J=7.8 Hz, 1H), 7.06 (d, J=7.0 Hz, 1H), 6.88 (d, J=7.6 Hz, 1H), 3.64 (s,2H, CH₂).

MS +ve APCI m/z 211 [M⁺+1].

5-(2-Oxo-2,3-dihydro-1H-indol-4-yl)-nicotinic Acid

Palladium catalyst Pd(PPh₃)₄ (693 mg, 0.6 mmol) and freshly preparedaqueous sodium hydroxide (3.2 g in 29 mL of water) were added to amixture of 4-(4,4,5,5-tetramethyl[1,3,2]-dioxaborolan-2-yl)-1H-indole(4.9 g, 20.2 mmol) and 5-bromonicotinic acid (4.04 g, 20 mmol) in THF(70 mL). The system was degassed and then purged three times withnitrogen. The mixture was refluxed under nitrogen for 6 hours. It wasthen cooled to room temperature and ethyl acetate (400 mL) added. Theorganic layer was separated and washed twice with 2N NaOH solution. Theaqueous layer and hydroxide washes were combined, washed withdichloromethane and then acidified with 6N HCl. The resultingprecipitate was collected by vacuum filtration, washed with water anddried to give 2.3 g (48%) 5-(1H-indol-4-yl)-nicotinic acid.

¹H NMR (360 MHz, DMSO-d₆) δ 11.45 (br s, 1H, NH), 9.06 (m, 2H), 8.49 (m,1H), 7.45 (m, 2H), 7.22 (m, 2H), 6.54 (s, 1H).

MS +ve APCI m/z 239 [M⁺+1].

Pyridinium tribromide (90% (Aldrich), 9.8 g, 27.6 mmol) was addedportion-wise over 10 minutes to a suspension of5-(1H-indol-4-yl)-nicotinic acid (2.2 g, 9.2 mmol) in2-methyl-2-propanol (54 mL), ethanol (36 mL) and acetic acid (108 mL).The mixture was stirred at room temperature for 2 hours and then aceticacid (10 mL) was added. After stirring for an additional hour, water(0.5 mL) and zinc dust (6 g, 92 mmol) were added and stirring wascontinued for another hour. Residual zinc dust was filtered and washedwith methanol. The filtrate was concentrated and the syrupy residue wasstirred in water (300 mL) overnight. The solid which formed wasfiltered, washed with water to remove zinc and pyridine salts and driedunder high vacuum to give 2.3 g (98%) of5-(2-oxo-2,3-dihydro-1H-indol-4-yl)-nicotinic acid.

¹H NMR (400 MHz, DMSO-d₆) δ 10.53 (s, 1H, NH), 9.08 (s, 1H), 8.92 (s,1H), 8.35 (s, 1H), 7.32 (t, J=7.5 Hz, 1H), 7.09 (d, J=7.5 Hz, 1H), 6.89(d, J=7.5 Hz, 1H), 3.65 (s, 2H, CH₂).

MS −ve APCI m/z 253 [M⁺−1].

4-(2-amino-pyrimidin-5-yl)-1,3-dihydro-indol-2-one

Pd(PPh₃)₄ (285 mg, 0.25 mmol) and a freshly prepared sodium hydroxidesolution (985 mg in 12 mL of water) were added to a mixture of4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-1H-indole (2 g, 8.21mmol) and 2-amino-5-bromopyrimidine (1.71 g, 9.85 mmol) in THF (30 mL).The reaction mixture was degassed and then purged three times withnitrogen and then refluxed overnight with stirring under nitrogen. Thereaction mixture was cooled to room temperature and then diluted withethyl acetate (300 mL). The organic layer was separated, washed withbrine, dried over anhydrous Na₂SO₄ and concentrated. The residue waspurified by column chromatography to give 1.4 g (82%) of5-(1H-indol-4-yl)-pyrimidin-2-ylamine as a white solid.

MS +ve APCI m/z 211 [M⁺+1].

Pyridinium tribromide (90% purity from Aldrich, 7.1 g, 19.98 mmol) wasadded in portions over 10 minutes to a suspension of5-(1H-indol-4-yl)-pyrimidin-2-ylamine (1.4 g, 6.66 mmol) in2-methyl-2-propanol (50 mL), ethanol (30 mL) and acetic acid (18 mL).The mixture was stirred at room temperature for 2 hours, and then aceticacid (72 mL), water (1 mL) and zinc (6.5 g, 100 mmol) were added.Stirring was continued for 1.5 hours. The unreacted zinc dust wasfiltered off and washed with methanol. The filtrate was concentrated andthe syrupy residue was suspended in water (100 mL) overnight. The solidwhich formed was filtered off. The filtrate was basified with aqueoussodium bicarbonate and extracted with ethanol:dichloromethane (5:95) togive 4-(2-amino-pyrimidin-5-yl)-1,3-dihydro-indol-2-one. ¹H NMR (360MHz, DMSO-d₆) δ 10.46 (s, 1H, NH), 8.47 (s, 2H), 7.24 (t, J=7.6 Hz, 1H),6.99 (d, J=7.6 Hz, 1H), 6.79 (d, J=7.6 Hz, 1H), 6.78 (s, 2H, NH₂), 3.64(s, 2H)

MS +ve APCI m/z 227 [M⁺+1].

General Amidation Procedure

A mixture of a carboxylic acid pyrrole aldehyde (1 equiv.), anappropriately substituted amine (1.2 equiv.), 1-hydroxybenzotriazole(HOBt, 1.2 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDAC, 1.2 equiv.) were dissolved in sufficientN,N-dimethylformamide (DMF) to make a 0.4 M solution. The mixture isstirred overnight at room temperature and then diluted withdichloromethane, washed with aqueous sodium bicarbonate, dried andconcentrated to give the desired amide.

Utilizing the 4-heteroarylindolinones described above and following theamidation procedures following compounds of Formula (I) were prepared.

Example 13-[3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-4-yl-1,3-dihydroindol-2-one

A mixture of 4-pyridin-4-yl-1,3-dihydroindol-2-one (50 mg, 0.24 mmol),3,5-dimethyl-4-(4-methyl-piperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde(59 mg, 0.24 mmol) and piperidine (0.1 mL) in ethanol (1 mL) was heatedat 60° C. for 5 hours. The reaction was concentrated and the residue wascolumn chromatographed to give the title compound.

¹H NMR (300 MHz, DMSO-d₆) δ 13.42 (br s, 1H, NH), 11.09 (br s, 1H, NH),8.72 (d, J=5.5 Hz, 2H), 7.49 (d, J=5.5 Hz, 2H), 7.21 (t, J=7.7 Hz, 1H),6.96 (d, J=7.7 Hz, 1H), 6.79 (d, J=7.7 Hz, 1H), 6.72 (s, 1H, H-vinyl),3.35 (m, 4H, 2×CH₂), 2.26 (m, 4H, 2×CH₂), 2.23 (s, 3H, CH₃), 2.18 (s,3H, CH₃), 1.54 (s, 3H, CH₃).

MS m/z 442 [M⁺+1].

Example 23-(5-Methyl-3H-imidazol-4-ylmethylene)-4-pyridin-4-yl-1,3-dihydroindol-2-one

A mixture of 4-pyridin-4-yl-1,3-dihydroindol-2-one (50 mg, 0.24 mmol),5-methyl-3H-imidazole-4-carbaldehyde (24.4 mg, 0.24 mmol) and piperidine(1 drop) in ethanol (2 mL) was stirred at room temperature for 2 days.The precipitate which formed was filtered. Crystals which formed in thefiltrate were isolated, washed with ethanol and dried to give 7.8 mg ofthe title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.55 (br s, 1H, NH), 11.13 (br s, 1H, NH),8.75 (d, J=6.0 Hz, 2H), 7.85 (s, 1H), 7.50 (d, J=6.0 Hz, 2H), 7.27 (t,J=7.7 Hz, 1H), 6.97 (d, J=7.7 Hz, 1H), 6.83 (d, J=7.7 Hz, 1H), 6.76 (s,1H, H-vinyl), 1.78 (m, 3H, CH₃)

MS m/z 303 [M⁺+1].

Example 31-(2-Oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-6,7-dihydro-2H-pyrano[3,4-c]pyrrol-4-one

A mixture of 4-pyridin-4-yl-1,3-dihydroindol-2-one (50 mg, 0.24 mmol),4-oxo-2,4,6,7-tetrahydropyrano[3,4-c]pyrrole-1-carbaldehyde (39.2 mg,0.24 mmol) and piperidine (1 drop) in ethanol (2 mL) was stirred at roomtemperature for 48 hours. The precipitate which formed was collected byvacuum filtration, washed with ethanol and dried to give 15 mg (17%) ofthe title compound.

¹HNMR (360 MHz, DMSO-d₆) δ 8.74 (d, J=6.1 Hz, 2H), 7.83 (s, 1H), 7.48(d, J=6.1 Hz, 2H), 7.28 (t, J=7.7 Hz, 1H), 6.99 (d, J=7.7 Hz, 1H), 6.84(d, J=7.7 Hz, 1H), 6.66 (s, 1H), 4.34 (t, J=5.7 Hz, 2H, CH₂), 2.29 (t,J=5.7 Hz, 2H, CH₂).

MS m/z 358 [M⁺+1].

Example 43-[3-Methyl-4-(piperidine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-4-yl-1,3-dihydroindol-2-one

A mixture of 4-pyridin-4-yl-1,3-dihydroindol-2-one (60 mg, 0.3 mmol),3-methyl-4-(piperidine-1-carbonyl)-1H-pyrrole-2-carbaldehyde (75 mg,0.34 mmol) and piperidine (0.14 mL) in ethanol (2 mL) was stirred at100° C. overnight. The reaction was column chromatographed (2-3%methanol in dichloromethane) to give 20 mg (17%) of the title compoundas a yellow orange solid.

¹H NMR (300 MHz, DMSO-d₆) δ 13.49 (br s, 1H, NH), 11.16 (br s, 1H, NH),8.73 (d, J=6.1 Hz, 2H), 7.50 (d, J 6.1 Hz, 2H), 7.37 (d, J=3.1 Hz, 1H),7.24 (t, 1H), 6.96 (d, 1H), 6.80 (d, 1H), 6.79 (s, 1H, H-vinyl), 3.40 (m4H, 2×CH₂), 1.6 (s, 3H, CH₃), 1.56 (m, 2H, CH₂), 1.44 (m, 4H, 2×CH₂).

MS m/z 413 [M⁺+1].

Example 5 3-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-4-pyridin-4-yl-1,3-dihydroindol-2-one

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with3,5-dimethyl-1H-pyrrole-2-carbaldehyde to give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.22 (br s, 1H, NH), 10.99 (br s, 1H, NH),8.73 (d, J=5.7 Hz, 2H), 7.48 (d, J=5.7 Hz, 2H), 7.18 (t, J=7.6 Hz, 1H),6.96 (d, J=7.6 Hz, 1H), 6.78 (d, J=7.6 Hz, 1H), 6.72 (s, 1H, H-vinyl),5.92 (s, 1H), 2.27 (s, 3H, CH₃), 1.61 (s, 3H, CH₃).

MS +ve APCI m/z 316 [M⁺+1].

Example 63-[2-(2-Oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionicAcid

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with3-(2-formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-propionic acid to give thetitle compound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.24 (br s, 1H, NH), 12.0 (br s, 1H, COOH),10.98 (br s, 1H, NH), 8.68 (d, J=5.9 Hz, 2H), 7.48 (d, J=5.9 Hz, 2H),7.17 (t, J=7.8 Hz, 1H), 6.95 (d, J=7.8 Hz, 1H), 6.84 (s, 1H, H-vinyl),6.75 (d, J=7.8 Hz, 1H), 2.63 (t, 2H, CH₂), 2.36 (t, 2H, CH₂), 2.16 (t,2H, CH₂), 2.01 (t, 2H, CH₂), 1.64-1.73 (m, 4H).

MS +ve APCI m/z 414 [M⁺+1].

Example 73-[5-Methyl-2-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionicAcid

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with3-(2-formyl-5-methyl-1H-pyrrol-3-yl)-propionic acid to give the titlecompound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.31 (br s, 1H, NH), 12.0 (br s, 1H, COOH),11.02 (br s, 1H, NH), 8.70 (d, J=5.8 Hz, 2H), 7.48 (d, J=5.8 Hz, 2H),7.19 (t, J=8 Hz, 1H), 6.96 (d, J=8 Hz, 1H), 6.82 (s, 1H, H-vinyl), 6.78(d, J=8 Hz, 1H), 5.96 (d, J=2.8 Hz, 2H), 2.28 (s, 3H, CH₃), 2.17 (m,4H).

MS +ve APCI m/z 374 [M⁺+1].

Example 83-[5-Ethyl-2-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionicAcid

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with3-(5-ethyl-2-formyl-1H-pyrrol-3-yl)-propionic acid to give the titlecompound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.39 (br s, 1H, NH), 10.98 (br s, 1H, NH),8.73 (d, J=5.2 Hz, 2H), 7.56 (d, J=5.2 Hz, 2H), 7.18 (t, J=7.7 Hz, 1H),6.97 (d, J=7.7 Hz, 1H), 6.79 (s, 1H, H-vinyl), 6.78 (d, J=7.7 Hz, 1H),6.01 (d, J=1.9 Hz, 1H), 2.63 (q, J=7.7 Hz, 2H, CH₂CH₃), 2.16 (m, 4H),1.18 (t, J=7.7 Hz, 3H, CH₂CH₃).

MS +ve APCI m/z 388 [M⁺+1].

Example 94-(2-Carboxyethyl)-2-methyl-5-(2-oxo-4-pyridin-4-yl-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid Ethyl Ester

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with4-(2-carboxyethyl)-5-formyl-2-methyl-1H-pyrrole-3-carboxylic acid ethylester to give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.82 (br s, 1H, NH), 11.8 (br s, 1H, COOH),11.21 (br s, 1H, NH), 8.68 (d, J=5.5 Hz, 2H), 7.49 (d, J=5.5 Hz, 2H),7.25 (t, J=7.6 Hz, 1H), 6.98 (d, J=7.6 Hz, 1H), 6.93 (s, 1H, H-vinyl),6.80 (d, J=7.6 Hz, 1H), 4.16 (q, J=7.2 Hz, 2H, OCH₂CH₃), 2.41 (t, J=7.4Hz, 2H), 2.14 (t, J=7.4 Hz, 2H), 1.25 (t, J=7.2 Hz, 3H, OCH₂CH₃).

MS +ve APCI m/z 446 [M⁺+1].

Example 103-[2,4-Dimethyl-5-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionicAcid

4-Pyridin-4-yl-1,3-dihydro-indol-2-one was condensed with3-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid to give thetitle compound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.28 (br s, 1H, NH), 12.0 (br s, 1H, COOH),10.96 (br s, 1H, NH), 8.73 (d, J=6 Hz, 2H), 7.48 (d, J=6 Hz, 2H), 7.17(t, J=7.6 Hz, 1H), 6.95 (d, J=7.6 Hz, 1H), 6.77 (d, J=7.6 Hz, 1H), 6.72(s, 1H, H-vinyl), 2.54 (t, J=7.6 Hz, 2H), 2.26 (m, 5H), 1.55 (s, 3H,CH₃).

MS +ve APCI m/z 388 [M⁺+1].

Example 11[2,4-Dimethyl-5-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-aceticAcid

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)-acetic acid to give the titlecompound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.31 (s, 1H, NH), 12.0 (br s, 1H, COOH),10.95 (s, 1H, NH), 8.72 (d, J=5.9 Hz, 2H), 7.51 (d, J=5.9 Hz, 2H), 7.17(t, J=7.7 Hz, 1H), 6.96 (d, J=7.7 Hz, 1H), 6.77 (d, J=7.6 Hz, 1H), 6.73(s, 1H, H-vinyl), 3.18 (s, 2H, CH₂), 2.25 (s, 3H, CH₃), 1.54 (s, 3H,CH₃)

MS +ve APCI m/z 374 [M⁺+1].

Example 123-(1H-Indol-2-ylmethylene)-4-pyridin-4-yl-1,3-dihydroindol-2-one

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with1H-indole-2-carbaldehyde to give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ 12.90 (br s, 1H, NH), 11.20 (br s, 1H, NH),8.77 (d, J=5.5 Hz, 2H), 7.56 (m, 4H), 7.33 (t, J=7.7 Hz, 1H), 7.25 (m,1H), 7.06 (m, 1H), 7.0 (d, J=7.7 Hz, 1H), 6.90 (s, 1H, H-vinyl), 6.86(d, J=7.7 Hz, 1H), 6.56 (s, 1H).

MS +ve APCI m/z 338 [M⁺+1].

Example 134-Pyridin-4-yl-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde to give the title compound.

Example 143-[5-(2-Morpholin-4-yl-ethoxy)-1H-indol-2-ylmethylene]-4-pyridin-4-yl-1,3-dihydroindol-2-one

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with(5-(2-morpholin-4-yl-ethoxy)-1H-indole-2-carbaldehyde to give the titlecompound.

¹H NMR (400 MHz, DMSO-d₆) δ 12.82 (s, 1H, NH), 11.24 (s, 1H, NH), 8.77(d, J=6.3 Hz, 2H), 7.55 (d, J=6.3 Hz, 2H), 7.48 (d, J=8.8 Hz, 1H), 7.31(t, J=7.7 Hz, 1H), 7.03 (d, J=2.5 Hz, 1H), 7.0 (d, J=7.7 Hz, 1H), 6.91(dd, J=2.5 & 8.8 Hz, 1H), 6.84 (m, 2H), 6.43 (s, 1H), 4.05 (t, J=5.8 Hz,2H), 3.57 (t, J=4.6 Hz, 4H), 2.68 (t, J=5.8 Hz, 2H), 2.47 (br t, 4H).

MS +ve APCI m/z 467 [M⁺+1].

Example 154-Methyl-5-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicAcid

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid to give the titlecompound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.67 (br s, 1H, NH), 12.90 (br s, 1H,COOH), 11.22 (br s, 1H, NH), 8.75 (d, J=5.8 Hz, 2H), 7.52 (d, J=5.8 Hz,2H), 7.30 (t, J=7.7 Hz, 1H), 7.0 (d, J=7.7 Hz, 1H), 6.78 (s, 1H,H-vinyl), 6.61 (d, J=2.5 Hz, 1H), 1.64 (s, 3H, CH₃).

MS −ve APCI m/z 344 [M⁺−1].

Example 165-Methyl-2-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid to give the titlecompound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.97 (s, 1H, NH), 12.08 (v br s, 1H, COOH),11.26 (br s, 1H, NH), 8.63 (d, J=6 Hz, 2H), 8.02 (s, 1H, H-vinyl), 7.42(d, J=6 Hz, 2H), 7.26 (t, J=7.7 Hz, 1H), 6.98 (d, J=7.7 Hz, 1H), 6.78(d, J=7.6 Hz, 1H), 6.39 (d, J=2.3 Hz, 1H), 2.31 (s, 3H, CH₃).

MS −ve APCI m/z 344 [M⁺−1].

Example 173-[3-(3-Morpholin-4-yl-propyl)-4,5,6,7-tetrahydro-1H-indol-2-ylmethylene]-4-pyridin-4-yl-1,3-dihydroindol-2-one

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with3-(3-morpholin-4-yl-propyl)-4,5,6,7-tetrahydro-1H-indole-2-carbaldehydeto give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.22 (s, 1H, NH), 10.97 (s, 1H, NH), 8.72(d, J=5.8 Hz, 2H), 7.50 (d, J=5.9 Hz, 2H), 7.17 (t, J=7.7 Hz, 1H), 6.95(d, J=7.7 Hz, 1H), 6.81 (s, 1H, H-vinyl), 6.75 (d, J=7.7 Hz, 1H), 3.55(m, 4H), 2.64 (t, 2H), 2.34 (t, 2H), 2.29 (br s, 4H), 2.04 (t, 2H), 1.90(t, 2H), 1.65-1.73 (m, 4H), 1.22 (m, 2H).

MS +ve APCI m/z 469 [M⁺+1].

Example 182,4-Dimethyl-5-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidene-methyl)-1H-pyrrole-3-carboxylicacid (2-diethylaminoethyl)amide

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)-amide to give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.46 (s, 1H, NH), 11.10 (br s, 1H, NH),8.74 (d, J=5.9 Hz, 2H), 7.50 (d, J=5.9 Hz, 2H), 7.35 (t, 1H, CONH), 7.22(t, J=7.7 Hz, 1H), 6.98 (d, J=7.7 Hz, 1H), 6.81 (d, J=7.6 Hz, 1H), 6.75(s, 1H, H-vinyl), 3.23 (q, J=6.8 Hz, 2H, NCH₂CH₃), 2.45-2.5 (m, 6H),2.39 (s, 3H, CH₃), 1.69 (s, 3H, CH₃), 0.95 (t, J=6.8 Hz, 6H,N(CH₂CH₃)₂).

MS −ve APCI m/z 456 [M⁺−1].

Example 193-[3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-4-yl-1,3-dihydroindol-2-one

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehydeto give the title compound.

MS −ve APCI m/z 440 [M⁺−1].

Example 203-[3-Methyl-5-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-4-yl-1,3-dihydroindol-2-one

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with3-methyl-5-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde togive the title compound.

Example 213-(2-Oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylicAcid Ethyl Ester

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with 3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethyl ester to give thetitle compound.

Example 222-Methyl-5-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidene-methyl)-4-phenyl-1H-pyrrole-3-carboxylicAcid pyridin-4-ylamide

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acidpyridin-4-ylamide to give the title compound.

Example 233-(5-Methylthiophen-2-ylmethylene)-4-pyridin-4-yl-1,3-dihydroindol-2-one

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with5-methylthiophene-2-carbaldehyde to give the title compound.

Example 243-(4-Morpholin-4-yl-benzylidene)-4-pyridin-4-yl-1,3-dihydroindol-2-one

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with4-morpholin-4-yl-benzaldehyde to give the title compound.

Example 254-[4-(2-Oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-phenyl]-piperazine-1-carbaldehyde

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with4-(4-formylphenyl)-piperazine-1-carbaldehyde to give the title compound.

Example 264-(2-Carboxyethyl)-3-methyl-5-(2-oxo-4-pyridin-4-yl-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicAcid Ethyl Ester

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with4-(2-carboxyethyl)-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acid ethylester to give the title compound.

Example 274-(2-Hydroxyethyl)-5-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with5-formyl-4-(2-hydroxyethyl)-1H-pyrrole-3-carboxylic acid to give thetitle compound.

Example 284-(4-Methoxyphenyl)-5-(2-oxo-4-pyridin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid Ethyl Ester

4-Pyridin-4-yl-1,3-dihydroindol-2-one was condensed with5-formyl-4-(4-methoxyphenyl)-1H-pyrrole-3-carboxylic acid ethyl ester togive the title compound.

Example 293-(5-Methyl-3H-imidazol-4-ylmethylene)-4-piperidin-4-yl-1,3-dihydroindol-2-one,Acetic Acid Salt

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (124 mg, 0.57 mmol)and 5-methyl-3H-imidazole-4-carbaldehyde (62.8 mg, 0.57 mmol) in ethanol(3 mL) was stirred at room temperature for 2 days. The reaction wasconcentrated and the residue was column chromatographed (reverse phase)to give 23 mg of the title compound as a yellow acetic acid salt.

¹H NMR (300 MHz, DMSO-d₆) δ 13.89 (br s, 1H, NH), 11.02 (br s, 1H, NH),7.90 (s, 1H), 7.52 (s, 1H), 7.15 (t, J 7.8 Hz, 1H), 6.93 (d, J=7.8 Hz,1H), 6.76 (d, J 7.8 Hz, 1H), 3.20 (m, 1H), 3.15 (m, 2H, CH₂), 2.73 (m,2H, CH₂), 1.86 (m, 3H, CH₃), 1.84 (m, 2H, CH₂), 1.62 (m, 2H, CH₂).

MS m/z 309 [M⁺+1].

Example 303-[3-Methyl-4-(piperidine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-piperidin-4-yl-1,3-dihydro-indol-2-one

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (86.5 mg, 0.4 mmol)and 3-methyl-4-(piperidine-1-carbonyl)-1H-pyrrole-2-carbaldehyde (88.1mg, 0.4 mmol) in ethanol (2 mL) was stirred at room temperature for 4days. The reaction was concentrated and the residue was columnchromatographed to give 52 mg (31%) of the title compound as a yellowsolid.

¹H NMR (300 MHz, DMSO-d₆) δ 13.80 (br s, 1H, NH), 11.0 (br s, 1H, NH),7.53 (s, 1H, H-vinyl), 7.40 (d, J=3.0 Hz, 1H), 7.13 (t, J=7.8 Hz, 1H),6.93 (d, J=7.8 Hz, 1H), 6.75 (d, J=7.8 Hz, 1H), 3.48 (m, 4H, 2×CH₂),3.20 (m, 1H), 3.10 (m, 2H, CH₂), 2.70 (m, 2H, CH₂), 2.29 (s, 3H, CH₃),1.84 (m, 2H, CH₂), 1.61 (m, 4H, 2×CH₂), 1.48 (m, 4H, 2×CH₂).

MS m/z 419 [M⁺+1].

Example 313-[3-Methyl-4-(morpholine-4-carbonyl)-1H-pyrrol-2-ylmethylene]-4-piperidin-4-yl-1,3-dihydroindol-2-one

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (86.5 mg, 0.4 mmol)and 3-methyl-4-(morpholine-4-carbonyl)-1H-pyrrole-2-carbaldehyde (88.9mg, 0.4 mmol) in ethanol (2 mL) was stirred at room temperature for 4days. The reaction was concentrated and the residue was columnchromatographed to give 54 mg (32%) of the title compound as a yellowsolid.

¹H NMR (360 MHz, DMSO-d₆) δ 13.82 (br s, 1H, NH), 10.97 (br s, 1H, NH),7.54 (s, 1H, H-vinyl), 7.45 (d, J=3.0 Hz, 1H), 7.14 (t, J=7.8 Hz, 1H),6.94 (d, J=7.8 Hz, 1H), 6.76 (d, J=7.8 Hz, 1H), 3.58 (m, 4H, 2×CH₂),3.53 (m, 4H, 2×CH₂), 3.20 (m, 1H), 3.12 (m, 2H, CH₂), 2.73 (m, 2H, CH₂),1.86 (m, 2H, CH₂), 1.65 (m, 2H, CH₂).

MS m/z 421 [M⁺+1].

Example 321-(2-Oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-6,7-dihydro-2H-pyrano[3,4-c]pyrrol-4-one

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (117 mg, 0.54mmol), 4-oxo-2,4,6,7-tetrahydropyrano[3,4-c]pyrrole-1-carbaldehyde (89.3mg, 0.54 mmol) and piperidine (1 drop) in ethanol (3 mL) was stirred atroom temperature for 2 days. The precipitate was collected by vacuumfiltration, washed with ethanol and dried to give 54.1 mg (28%) of thetitle compound as a yellow solid.

¹HNMR (360 MHz, DMSO-d₆) δ 14.0 (br s, 1H, NH), 11.06 (br s, 1H, NH),7.90 (d, J=2.8 Hz, 1H), 7.44 (s, 1H, H-vinyl), 7.16 (t, J=8 Hz, 1H),6.95 (d, J=8 Hz, 1H), 6.77 (d, J=8 Hz, 1H), 4.34 (t, J=65.7 Hz, 2H,CH₂), 2.29 (t, J=5.7 Hz, 2H, CH₂).

MS m/z 364 [M⁺+1].

Example 331-(2-Oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-2,5,6,7-tetrahydropyrrolo[3,4-c]pyridin-4-one

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (79 mg, 0.36 mmol),4-oxo-4,5,6,7-tetrahydro-2H-pyrrolo[3,4-c]pyridine-1-carbaldehyde (50mg, 0.3 mmol) and piperidine (1 drop) in ethanol (2 mL) was stirred atroom temperature for 4 days. The precipitate was collected by vacuumfiltration, washed with ethanol followed by 0.1% of acetic acid in waterand dried to give 30 mg (23%) of the title compound.

¹HNMR (360 MHz, DMSO-d₆) δ 13.8 (br s, 1H, NH), 11.0 (br s, 1H, NH),7.66 (d, J=2.9 Hz, 1H), 7.45 (s, 1H, H-vinyl), 7.31 (br s, 1H, NH), 7.15(t, J=8 Hz, 1H), 6.93 (d, J=8 Hz, 1H), 6.77 (d, J=8 Hz, 1H), 3.44 (m,2H, CH₂), 3.22 (m, 1H), 3.14 (m, 2H), 2.90 (t, 2H, CH₂), 2.76 (t, 2H,CH₂), 1.85 (m, 2H), 1.63 (m, 2H).

MS APCI +ve 363 [M⁺+1].

Example 345-Methyl-1-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidene-methyl)-2,5,6,7-tetrahydro-pyrrolo[3,4-c]pyridin-4-one

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (58.4 mg, 0.27mmol),5-methyl-4-oxo-4,5,6,7-tetrahydro-2H-pyrrolo[3,4-c]pyridine-1-carbaldehyde(48 mg, 0.27 mmol) and piperidine (1 drop) in ethanol (1 mL) was stirredat room temperature for 7 days. The precipitate was collected by vacuumfiltration, washed with ethanol and dried to give 58 mg (57%) of thetitle compound as a yellow solid.

¹HNMR (360 MHz, DMSO-d₆) δ 13.81 (br s, 1H, NH), 11.03 (br s, 1H, NH),8.84 (s, 1H, H-vinyl), 7.16 (t, J=7.7 Hz, 1H), 7.12 (d, J=2.2 Hz, 1H),6.96 (d, J=7.7 Hz, 1H), 6.75 (d, J=7.7 Hz, 1H), 3.54 (t, 2H), 3.3 (m,1H), 3.04 (m, 4H), 3.01 (s, 3H, CH₃), 2.80 (t, 2H), 1.85 (m, 2H), 1.6(m, 2H).

MS m/z 377 [M⁺+1].

Example 353-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-4-piperidin-4-yl-1,3-dihydroindol-2-one

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol),3,5-dimethyl-1H-pyrrole-2-carbaldehyde (29 mg, 0.23 mmol) andpyrrolidine (0.006 mL) in ethanol (0.5 mL) was refluxed for 2 hours at90° C. The reaction was cooled and the precipitate which formed wascollected by vacuum filtration, washed with ethanol and dried to givethe title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.52 (br s, 1H, NH), 10.82 (br s, 1H, NH),7.48 (s, 1H, H-vinyl), 7.07 (t, J=7.8 Hz, 1H), 6.91 (d, J=7.8 Hz, 1H),6.75 (d, J=7.8 Hz, 1H), 6.03 (d, J=2.2 Hz, 1H), 3.16 (m, 1H), 3.1 (m,2H), 2.69 (m, 2H), 2.32 (s, 3H, CH₃), 2.28 (s, 3H, CH₃), 1.83 (m, 2H),1.60 (m, 2H).

MS +ve APCI m/z 322 [M⁺+1].

Example 363-[2-(2-Oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-1H-indol-3-yl]-propionicAcid

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol),3-(2-formyl-4,5,6,7-tetrahydro-1H-indol-3-yl)-propionic acid (57 mg,0.23 mmol) and pyrrolidine (0.3 mL) in ethanol (0.5 mL) was heated toreflux for 2 hours. Acetic acid (0.05 mL) was added to the reaction andheating was continued for another 10 minutes. The reaction was cooledand the precipitate which formed was collected by vacuum filtration,washed with ethanol and dried to give the title compound.

MS +ve APCI m/z 420 [M⁺+1].

Example 373-[5-Methyl-2-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionicAcid

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 3-(2-formyl-5-methyl-1H-pyrrol-3-yl)-propionic acid (42 mg, 0.23mmol) to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 10.85 (br s, 1H), 7.58 (s, 1H, H-vinyl),7.08 (t, J=7.7 Hz, 1H), 6.90 (d, J=7.7 Hz, 1H), 6.76 (d, J=7.7 Hz, 1H),6.06 (d, J=1.8 Hz, 1H), 3.19 (m, 2H), 2.86-3.0 (m, 5H), 2.42 (m, 2H),2.33 (s, 3H, CH₃), 1.91 (m, 2H), 1.73 (m, 2H).

MS +ve APCI m/z 380 [M⁺+1].

Example 383-[5-Ethyl-2-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionicAcid

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 3-(5-ethyl-2-formyl-1H-pyrrol-3-yl)-propionic acid (0.23 mmol) togive the title compound.

MS +ve APCI m/z 394 [M⁺+1].

Example 394-(2-Carboxyethyl)-2-methyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid Ethyl Ester

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 4-(2-carboxyethyl)-5-formyl-2-methyl-1H-pyrrole-3-carboxylic acidethyl ester (56 mg, 0.23 mmol) to give the title compound.

MS +ve APCI m/z 452 [M⁺+1].

Example 403-[2,4-Dimethyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionicAcid

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 3-(5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)-propionic acid (43 mg,0.23 mmol) to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.58 (s, 1H, NH), 10.80 (s, 1H, NH), 7.47(s, 1H, H-vinyl), 7.06 (t, J=7.7 Hz, 1H), 6.89 (d, J=7.7 Hz, 1H), 6.75(d, J=7.7 Hz, 1H), 3.22 (m, 1H), 3.15 (m, 2H), 2.77 (m, 2H), 2.64 (m,2H), 2.3 (m, 2H), 2.29 (s, 3H, CH₃), 2.23 (s, 3H, CH₃), 1.87 (m, 2H),1.68 (m, 2H).

MS +ve APCI m/z 394 [M⁺+1].

Example 41[2,4-Dimethyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrol-3-yl]-aceticAcid

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith (5-formyl-2,4-dimethyl-1H-pyrrol-3-yl)-acetic acid (40 mg, 0.23mmol) to give the title compound.

MS +ve APCI m/z 380 [M⁺+1].

Example 423-(1H-Indol-2-ylmethylene)-4-piperidin-4-yl-1,3-dihydroindol-2-one

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol)was condensed with 1H-indole-2-carbaldehyde (32 mg, 0.23 mmol) to givethe title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.12 (s, 1H, NH), 7.70 (s, 1H, H-vinyl),7.66 (d, J 8.1 Hz, 1H), 7.60 (d, J=8.1 Hz, 1H), 7.28 (t, 1H), 7.20 (m,2H), 7.08 (t, J=7.7 Hz, 1H), 6.95 (d, J=7.7 Hz, 1H), 6.80 (d, J=7.7 Hz,1H), 3.25 (m, 3H), 3.0 (m, 2H), 1.93 (m, 2H), 1.76 (m, 2H).

MS +ve APCI m/z 344 [M⁺+1].

Example 434-Piperidin-4-yl-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde (33 mg, 0.23 mmol) togive the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.25 (s, 1H, NH), 10.84 (s, 1H, NH), 7.43(s, 1H, H-vinyl), 7.11 (t, J=7.7 Hz, 1H), 6.87 (d, J=7.7 Hz, 1H), 6.76(d, J=7.7 Hz, 1H), 6.58 (s, 1H), 3.4 (m, 3H), 2.92 (m, 2H), 2.71 (m,2H), 1.7-1.9 (m, 1OH).

MS −ve APCI 346 [M⁺−1].

Example 443-[5-(2-Morpholin-4-yl-ethoxy)-1H-indol-2-ylmethylene]-4-piperidin-4-yl-1,3-dihydroindol-2-one

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith (5-(2-morpholin-4-yl-ethoxy)-1H-indole-2-carbaldehyde (60 mg, 0.23mmol) to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.04 (s, 1H, NH), 11.1 (br s, 1H, NH), 7.66(s, 1H, H-vinyl), 7.51 (d, J=8.7 Hz, 1H), 7.2 (t, J=7.8 Hz, 1H), 7.12(br s, 1H), 7.06 (br s, 1H), 6.94 (m, 2H), 6.79 (d, J=7.8 Hz, 1H), 4.10(t, 2H, CH₂), 3.59 (t, 4H, 2×CH₂), 3.23 (m, 3H), 2.99 (m, 2H), 2.71 (t,2H, CH₂), 2.5 (m, 4H, 2×CH₂), 1.93 (m, 2H), 1.76 (m, 2H).

MS +ve APCI m/z 473 [M⁺+1].

Example 454-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicAcid

4-Piperidin-4-yl-1,3-dihydro-indol-2-one (45 mg, 0.2 mmol) was condensedwith 5-formyl-4-methyl-1H-pyrrole-2-carboxylic acid (34 mg, 0.23 mmol)to give the title compound.

MS +ve APCI m/z 352 [M⁺+1].

Example 465-Methyl-2-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid

4-Piperidin-4-yl-1,3-dihydroindol-2-onem (45 mg, 0.2 mmol) was condensedwith 2-formyl-5-methyl-1H-pyrrole-3-carboxylic acid (34 mg, 0.23 mmol)to give the title compound.

MS +APCI m/z 352 [M⁺+1].

Example 473-[3-(3-Morpholin-4-yl-propyl)-4,5,6,7-tetrahydro-1H-indol-2-ylmethylene]-4-piperidin-4-yl-1,3-dihydroindol-2-one

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith3-(3-morpholin-4-yl-propyl)-4,5,6,7-tetrahydro-1H-indole-2-carbaldehyde(61 mg, 0.23 mmol) to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.51 (s, 1H, NH), 10.81 (s, 1H, NH), 7.49(s, 1H, H-vinyl), 7.08 (t, J=7.5 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.76(d, J=7.5 Hz, 1H), 3.51 (m, 4H), 3.25 (m, 3H), 2.81 (t, 2H), 2.68 (m,2H), 2.62 (m, 2H), 2.44 (m, 2H), 2.28 (m, 6H), 1.64-1.92 (m, 10H).

MS +ve APCI m/z 475 [M⁺+1].

Example 482,4-Dimethyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid (2-diethylamino-ethyl)-amide

4-Piperidin-4-yl-1,3-dihydroindol-2-one was condensed with5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)-amide to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.79 (s, 1H, NH), 11.0 (br s, 1H, NH), 7.51(s, 1H, H-vinyl), 7.44 (t, J=5.6 Hz, 1H, CONHCH₂), 7.11 (t, J=7.7 Hz,1H), 6.93 (d, J=7.7 Hz, 1H), 6.77 (d, J=7.7 Hz, 1H), 3.3 (m, 4H), 2.72(m, 2H), 2.52 (q, J=7.2 Hz, 4H, N(CH₂CH₃)₂), 2.43 (s, 3H, CH₃), 2.37 (s,3H, CH₃), 1.85 (m, 2H), 1.64 (m, 2H), 0.97 (t, J=7.2 Hz, 6H, N(CH₂CH₃)₂.

MS +ve APCI m/z 464 [M⁺+1].

Example 493-[3-Methyl-5-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-piperidin-4-yl-1,3-dihydroindol-2-one

4-Piperidin-4-yl-1,3-dihydro-indol-2-one (45 mg, 0.2 mmol) was condensedwith3-methyl-5-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde (52mg, 0.23 mmol) to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.80 (s, 1H, NH), 7.51 (s, 1H, H-vinyl),7.16 (t, 1H), 6.95 (d, 1H), 6.77 (d, 1H), 6.60 (d, 1H), 3.67 (m, 4H),3.1-3.25 (m, 3H), 2.73 (m, 2H), 2.36 (m, 4H), 2.21 (s, 3H, CH₃), 2.19(s, 3H, CH₃), 1.85 (m, 2H), 1.61 (m, 2H).

MS +ve APCI m/z 434 [M⁺+1].

Example 503-(2-Oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylicAcid Ethyl Ester

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 3-formyl-4,5,6,7-tetrahydro-2H-isoindole-1-carboxylic acid ethylester (48 mg, 0.23 mmol) to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.89 (s, 1H, NH), 11.1 (br s, 1H, NH), 7.40(s, 1H, H-vinyl), 7.19 (t, J=7.7 Hz, 1H), 6.94 (d, J=7.7 Hz, 1H), 6.79(d, J=7.7 Hz, 1H), 4.28 (q, J=7.1 Hz, 2H, OCH₂CH₃), 3.25 (m, 3H), 2.87(m, 2H), 2.72 (m, 4H), 1.90 (m, 2H), 1.73 (m, 6H), 1.31 (t, J=7.1 Hz,3H, OCH₂CH₃).

MS +ve APCI m/z 420 [M⁺+1].

Example 512-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-4-phenyl-1H-pyrrole-3-carboxylicAcid pyridin-4-ylamide

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 5-formyl-2-methyl-4-phenyl-1H-pyrrole-3-carboxylic acidpyridin-4-ylamide (67 mg, 0.23 mmol) to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 14.15 (s, 1H, NH), 11.05 (br s, 1H, NH),9.78 (s, 1H), 8.36 (d, J=6.2 Hz, 2H), 7.44 (m, 9H), 7.12 (t, J=7.8 Hz,1H), 6.85 (d, J=7.8 Hz, 1H), 6.77 (d, J=7.8 Hz, 1H), 2.71 (m, 3H), 2.54(s, 3H, CH₃), 1.76 (m, 2H), 1.58 (m, 2H).

MS +ve APCI m/z 504 [M⁺+1].

Example 523-(5-Methylthiophen-2-ylmethylene)-4-piperidin-4-yl-1,3-dihydro-indol-2-one

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 5-methylthiophene-2-carbaldehyde (28 mg, 0.23 mmol) to give thetitle compound.

MS +ve APCI m/z 325 [M⁺+1].

Example 533-(4-Morpholin-4-yl-benzylidene)-4-piperidin-4-yl-1,3-dihydro-indol-2-one

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 4-morpholin-4-yl-benzaldehyde (43 mg, 0.23 mmol) to give the titlecompound.

¹H NMR (360 MHz, DMSO-d₆) δ 10.48 (s, 1H, NH), 8.18 (d, J=8.9 Hz, 2H),7.52 (s, 1H, H-vinyl), 7.12 (t, J=8.0 Hz, 1H), 6.98 (d, J=8.9 Hz, 2H),6.87 (d, J=8.0 Hz, 1H), 6.67 (d, J=8.0 Hz, 1H), 3.73 (m, 4H), 3.35 (m,4H), 3.2 (m, 3H), 2.79 (m, 2H), 1.86 (m, 2H), 1.72 (m, 2H).

MS +ve APCI m/z 390 [M⁺+1].

Example 544-(2-Carboxyethyl)-3-methyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-2-carboxylicAcid Ethyl Ester

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 4-(2-carboxyethyl)-5-formyl-3-methyl-1H-pyrrole-2-carboxylic acidethyl ester (55 mg, 0.23 mmol) to give the title compound.

MS +ve APCI m/z 452 [M⁺+1].

Example 554-(2-Hydroxyethyl)-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one was condensed with5-formyl-4-(2-hydroxyethyl)-1H-pyrrole-3-carboxylic acid to give thetitle compound.

MS +ve APCI m/z 382 [M⁺+1].

Example 564-(4-Methoxyphenyl)-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid Ethyl Ester

4-Piperidin-4-yl-1,3-dihydroindol-2-one (45 mg, 0.2 mmol) was condensedwith 5-formyl-4-(4-methoxyphenyl)-1H-pyrrole-3-carboxylic acid ethylester (60 mg, 0.23 mmol) to give the title compound.

¹HNMR (360 MHz, DMSO-d₆) δ 14.13 (s, 1H, NH), 11.03 (br s, 1H, NH), 7.35(s, 1H, H-vinyl), 7.25 (d, J=8.7 Hz, 2H), 7.10 (t, J=7.8 Hz, 1H), 7.0(d, J=8.7 Hz, 2H), 6.84 (d, J=7.8 Hz, 1H), 6.74 (d, J=7.8 Hz, 1H), 4.02(q, J=7.0 Hz, 3H, OCH₂CH₃), 3.81 (s, 3 h, OCH₃), 2.75 (m, 3H), 1.83 (m,2H), 1.52 (m, 2H), 1.37 (m, 2H), 1.04 (t, J=7.0 Hz, 3H, OCH₂CH₃).

Example 574-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidene-methyl)-1H-pyrrole-3-carboxylicAcid Benzylamide

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (54 mg, 0.25 mmol),5-formyl-4-methyl-1H-pyrrole-3-carboxylic acid benzylamide (63.5 mg,0.2625 mmol) and piperidine (2 drops) in ethanol (0.5 mL) was stirred atroom temperature for 7 days. The precipitate which formed was collectedby vacuum filtration, washed with ethanol and dried to give the titlecompound.

¹HNMR (360 MHz, DMSO-d₆) δ 11.08 (br s, 1H, NH), 8.54 (t, 1H), 7.86 (d,1H), 7.50 (s, 1H), 7.32 (m, 5H), 7.18 (t, 1H), 6.91 (d, 1H), 6.82 (d,1H), 4.43 (d, 2H), 3.44 (m, 3H), 3.08 (t, 2H), 2.55 (s, 3H, CH₃), 2.06(m, 2H), 1.92 (m, 2H).

MS −ve APCI m/z 439 [M⁺−1].

Example 584-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid (pyridin-4-ylmethyl)-amide

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (54 mg, 0.25 mmol),5-formyl-4-methyl-1H-pyrrole-3-carboxylic acid(pyridin-4-ylmethyl)-amide (64 mg, 0.2625 mmol) and piperidine (2 drops)in ethanol (0.5 mL) was stirred at room temperature for 7 days. Theprecipitate which formed was collected by vacuum filtration, washed withethanol and dried to give the title compound.

¹HNMR (360 MHz, DMSO-d₆) δ 11.08 (br s, 1H, NH), 8.64 (t, 1H), 8.51 (d,2H), 7.88 (m, 1H), 7.51 (s, 1H), 7.31 (d, 2H), 7.19 (t, 1H), 6.92 (d,1H), 6.82 (d, 1H), 4.44 (m, 2H), 3.55 (m, 3H), 3.07(m, 2H), 2.54 (s, 3H,CH₃), 2.06 (m, 2H), 1.90 (m, 2H).

MS −ve APCI m/z 440 [M⁺−1].

Example 594-Methyl-5-(2-oxo-4-piperidin-4-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid [3-(2-oxo-pyrrolidin-1-yl)-propyl]-amide

A mixture of 4-piperidin-4-yl-1,3-dihydroindol-2-one (35.6 mg, 0.165mmol), 5-formyl-4-methyl-1H-pyrrole-3-carboxylic acid[3-(2-oxo-pyrrolidin-1-yl)propyl]-amide (48 mg, 0.173 mmol) andpiperidine (2 drops) in ethanol (0.5 mL) was stirred at room temperaturefor 7 days. The reaction was concentrated and ether was added to theresidue. The precipitate which formed was collected by vacuumfiltration, washed with ethanol and ether and dried to give the titlecompound.

¹HNMR (360 MHz, DMSO-d₆) δ 11.07 (s, 1H, NH), 8.05 (br s, 1H), 7.94 (t,1H, NH), 7.76 (d, 1H), 7.49 (s, 1H), 7.18 (t, 1H), 6.91 (d, 1H), 6.82(d, 1H), 3.47 (m, 3H), 3.35 (m, 2H), 3.15 (m, 6H), 2.53 (s, 3H, CH₃),2.22 (m, 2H), 2.08 (m, 2H), 1.93 (m, 2H), 1.67 (m, 2H).

MS −ve APCI m/z 474 [M⁺−1].

Example 602-Methyl-4-[3-(4-methylpiperazin-1-yl)-propyl]-5-(2-oxo-4-pyridin-2-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid Ethyl Ester

A mixture of 4-pyridin-2-yl-1,3-dihydroindol-2-one (31.5 mg, 0.15 mmol),5-formyl-2-methyl-4-[3-(4-methylpiperazin-1-yl)-propyl]-1H-pyrrole-3-carboxylicacid ethyl ester (48.2 mg, 0.15 mmol) and piperidine (0.1 mL) in ethanol(1 mL) was heated in a sealed tube at 70° C. for 6 hours. The reactionwas concentrated and the residue was re-crystallized from ethyl acetateand hexane to give the title compound.

¹HNMR (360 MHz, DMSO-d₆) δ 13.92 (s, 1H, NH), 11.2 (br s, 1H, NH), 8.74(d, J=4.4 Hz, 1H), 7.99 (dt, J=1.8 & 7.6 Hz, 1H), 7.61 (d, J=7.5 Hz,1H), 7.54 (m, 1H), 7.24 (t, J=7.6 Hz, 1H), 6.98 (m, 2H), 6.90 (d, J=7.2Hz, 1H), 4.17 (q, J=7.0 Hz, 2H, OCH₂CH₃), 2.50 (s, 3H, CH₃), 2.27 (m,10H), 2.11 (s, 3H, CH₃), 1.99 (br t, 2H), 1.3 (m, 2H), 1.26 (t, J=7.0Hz, 3H, OCH₂CH₃).

MS −ve APCI m/z 512 [M⁺−1].

Example 613-[3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-2-yl-1,3-dihydroindol-2-one

A mixture of 4-pyridin-2-yl-1,3-dihydroindol-2-one (31.5 mg, 0.15 mmol),3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde(37.4 mg, 0.15 mmol) and piperidine (0.1 mL) in ethanol (1 mL) washeated in a sealed tube at 70° C. for 6 hours. The reaction wasconcentrated and the residue was re-crystallized from ethyl acetate andhexane to give the title compound.

¹HNMR (360 MHz, DMSO-d₆) δ 13.53 (s, 1H, NH), 11.06 (br s, 1H, NH), 8.74(d, J=4.0 Hz, 1H), 7.99 (dt, J=1.7 & 7.7 Hz, 1H), 7.59 (d, J=7.7 Hz,1H), 7.50 (m, 1H), 7.22 (t, J=7.7 Hz, 1H), 6.97 (d, J=7.7 Hz, 1H), 6.90(d, J=7.7 Hz, 1H), 6.85 (s, 1H), 3.32 (s, 3H, CH₃), 2.24 (s, 8H), 2.16(s, 3H, CH₃), 1.64 (s, 3H, CH₃).

MS −ve APCI m/z 440 [M⁺−1].

Example 623-[3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-pyrimidin-5-yl-1,3-dihydroindol-2-one

4-Pyrimidin-5-yl-1,3-dihydroindol-2-one (53 mg, 0.25 mmol) was condensedwith 3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde(69 mg, 0.275 mmol) to give the title compound.

MS +ve APCI m/z 443 [M⁺+1].

Example 633-[3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-thiazol-2-yl-1,3-dihydroindol-2-one

4-Thiazol-2-yl-1,3dihydro-indol-2-one (54 mg, 0.25 mmol) was condensedwith3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde(69 mg, 0.275 mmol) to give the title compound.

MS +ve APCI m/z 448 [M⁺+1].

Example 642-Methyl-4-[3-(4-methylpiperazin-1-yl)propyl]-5-(2-oxo-4-pyrimidin-5-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid Ethyl Ester

4-Pyrimidin-5-yl-1,3-dihydroindol-2-one (53 mg, 0.25 mmol) was condensedwith5-formyl-2-methyl-4-[3-(4-methylpiperazin-1-yl)-propyl]-1H-pyrrole-3-carboxylicacid ethyl ester (88 mg, 0.275 mmol) to give the title compound.

MS +ve APCI m/z 515 [M⁺+1].

Example 652-Methyl-4-[3-(4-methylpiperazin-1-yl)propyl]-5-(2-oxo-4-thiazol-2-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid Ethyl Ester

4-Thiazol-2-yl-1,3-dihydroindol-2-one (54 mg, 0.25 mmol) was condensedwith5-formyl-2-methyl-4-[3-(4-methylpiperazin-1-yl)propyl]-1H-pyrrole-3-carboxylicacid ethyl ester (88 mg, 0.275 mmol) to give the title compound.

¹HNMR (360 MHz, DMSO-d₆) δ 13.96 (br s, 1H, NH), 11.22 (br s, 1H, NH),8.15 (s, 1H, H-vinyl), 8.09 (d, J=3.3 Hz, 1H), 8.02 (d, J=3.3 Hz, 1H),7.27 (t, J=7.7 Hz, 1H), 7.17 (d, J=7.7 Hz, 1H), 7.06 (d, J=7.7 Hz, 1H),4.20 (q, 2H), 3.43 (m, 2H), 2.62 (t, 2H), 2.53 (s, 3H, CH₃), 2.24 (m,8H), 2.09 (s, 3H, CH₃), 1.48 (m, 2H), 1.05 (t, 3H).

Example 665-[4-(6-Aminopyridin-3-yl)-2-oxo-1,2-dihydroindol-3-ylidene-methyl]-2-methyl-4-[3-(4-methylpiperazin-1-yl)propyl]-1H-pyrrole-3-carboxylicAcid Ethyl Ester

4-(6-Aminopyridin-3-yl)-1,3-dihydroindol-2-one (56 mg, 0.25 mmol) wascondensed with5-formyl-2-methyl-4-[3-(4-methyl-piperazin-1-yl)propyl]-1H-pyrrole-3-carboxylicacid ethyl ester (88 mg, 0.275 mmol) to give the title compound.

¹HNMR (360 MHz, DMSO-d₆) δ 13.84 (br s, 1H, NH), 11.06 (br s, 1H, NH),7.95 (d, 1H), 7.44 (dd, 1H), 7.24 (s, 1H), 7.17 (t, 1H), 6.88 (d, 1H),6.74 (d, 1H), 6.58 (d, 1H), 6.14 (d, 1H), 4.18 (q, 2H), 3.27 (s, 3H,CH₃), 2.40 (m, 2H), 2.25 (m, 8H), 2.10 (s, 3H, CH₃), 2.08 (m, 2H), 1.34(m, 2H), 1.27 (t, 3H).

MS −ve APCI m/z 527 [M⁺−1].

Example 674-(6-Aminopyridin-3-yl)-3-[3,5-dimethyl-4-(4-methylpiperazin-1-ylcarbonyl)-1H-pyrrol-2-ylmethylene]-1,3-dihydroindol-2-one

4-(6-Aminopyridin-3-yl)-1,3-dihydroindol-2-one (56 mg, 0.25 mmol) wascondensed with3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde(69 mg, 0.275 mmol) to give the title compound.

MS −ve APCI m/z 455 [M⁺−1].

Example 682-Methyl-4-[3-(4-methylpiperazin-1-yl)propyl]-5-(2-oxo-4-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid Ethyl Ester

A mixture of 4-pyridin-3-yl-1,3-dihydroindol-2-one (42 mg, 0.2 mmol),5-formyl-2-methyl-4-[3-(4-methylpiperazin-1-yl)-propyl]-1H-pyrrole-3-carboxylicacid ethyl ester (64 mg, 0.2 mmol) and piperidine (0.1 mL) in ethanolwas heated in a sealed tube at 70° C. for 5 hours. The reaction wasconcentrated and the residue was purified by column chromatography togive the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.84 (s, 1H, NH), 11.20 (s, 1H, NH), 8.75(dd, J=1.6 & 4.7 Hz, 1H), 8.68 (d, J=1.6 Hz, 1H), 7.93 (dt, 1H), 7.60(dd, 1H), 7.26 (t, J=7.6 Hz, 1H), 6.99 (d, J=7.6 Hz, 1H), 6.83 (d, J=7.6Hz, 1H), 6.80 (s, 1H, H-vinyl), 4.17 (q, J=7.1 Hz, 2H, OCH₂CH₃),2.4-2.75 (m, 11H), 2.38 (s, 3H, CH₃), 2.17 (m, 4H), 1.30 (m, 2H), 1.26(t, J=7.1 Hz, 3H, OCH₂CH₃).

MS −ve APCI m/z 512 [M⁺−1].

Example 693-[3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-4-pyridin-3-yl-1,3-dihydroindol-2-one

A mixture of 4-pyridin-3-yl-1,3-dihydroindol-2-one (42 mg, 0.2 mmol),3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde(50 mg, 0.2 mmol) and piperidine (0.1 mL) in ethanol was heated in asealed tube at 70° C. for 5 hours. The reaction was concentrated and theresidue was crystallized from ethyl acetate/hexane to give the titlecompound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.44 (s, 1H, NH), 11.08 (s, 1H, NH), 8.75(dd, 1H), 8.64 (d, 1H), 7.89 (br d, 1H), 7.58 (dd, 1H), 7.23 (t, J=7.7Hz, 1H), 6.98 (d, J=7.7 Hz, 1H), 6.83 (d, J=7.7 Hz, 1H), 6.66 (s, 1H,H-vinyl), 3.4 (m, 4H), 2.24 (br s, 7H), 2.16 (s, 3H, CH₃), 1.55 (s, 3H,CH₃).

MS −ve APCI m/z 440 [M⁺−1].

Example 705-(3-{4-Ethoxycarbonyl-5-methyl-3-[3-(4-methylpiperazin-1-yl)-propyl]-1H-pyrrol-2-ylmethylene}-2-oxo-2,3-dihydro-1H-indol-4-yl)-nicotinicAcid

A mixture of 5-(2-oxo-2,3-dihydro-1H-indol-4-yl)-nicotinic acid (51 mg,0.2 mmol),5-formyl-2-methyl-4-[3-(4-methyl-piperazin-1-yl)-propyl]-1H-pyrrole-3-carboxylicacid ethyl ester (64 mg, 0.2 mmol) and piperidine (0.2 mL) in ethanol (1mL) was heated in a sealed tube at 60° C. for 5 hours.

The reaction was acidified with 1N HCl and the resulted precipitate wascollected by vacuum filtration, washed with water and dried to give thetitle compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.88 (s, 1H, NH), 11.22 (s, 1H, NH), 9.18(d, J=2.1 Hz, 1H), 8.71 (d, J=2.1 Hz, 1H), 8.24 (m, 1H), 7.26 (t, J=7.6Hz, 1H), 6.99 (d, J=7.6 Hz, 1H), 6.84 (d, J=7.6 Hz, 1H), 6.77 (s, 1H,H-vinyl), 4.14 (q, J=7.4 Hz, 2H, OCH₂CH₃), 2.72 (m, 8H), 2.5 (s, 3H,CH₃), 2.38 (s, 3H, CH₃), 2.2-2.4 (m, 4H), 1.25 (m, 2H), 1.23 (t, J=7.4Hz, 3H, OCH₂CH₃).

MS +ve APLCI m/z 558 [M⁺+1].

Example 715-{3-[3,5-Dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H-indol-4-yl}-nicotinicAcid

A mixture of 5-(2-oxo-2,3-dihydro-1H-indol-4-yl)-nicotinic acid (381 mg,1.5 mmol),3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde(374 mg, 1.5 mmol) and piperidine (1 mL) in ethanol (5 mL) was heated ina sealed cube at 60° C. for 5 hours. The reaction was acidified with 1NHCl and the resulted precipitate was collected by vacuum filtration,washed with water and dried to give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.49 (s, 1H, NH), 11.61 (s, 1H, NH), 9.18(s, 1H), 8.88 (s, 1H), 8.30 (s, 1H), 7.26(m, 1H), 7.01 (d, J=7.4 Hz,1H), 6.89 (d, J=7.4 Hz, 1H), 6.66 (s, 1H, H-vinyl), 3.58 (br s, 4H),2.91 (br s, 4H), 2.61 (s, 3H, CH₃), 2.27 (s, 3H, CH₃), 1.55 (s, 3H,CH₃).

MS −ve APCI m/z 484 [M⁺−1].

Example 725-{3-[4-(2-Diethylaminoethylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-ylmethylene]-2-oxo-2,3-dihydro-1H-indol-4-yl}-nicotinicAcid

A mixture of 5-(2-oxo-2,3-dihydro-1H-indol-4-yl)-nicotinic acid (381 mg,1.5 mmol), 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)-amide (398 mg, 1.5 mmol) and piperidine (1 mL) inethanol (5 mL) was heated in a sealed tube at 60° C. for 10 hours. Thereaction was concentrated, the residue was dissolved in a mixture ofwater and methanol and then acidified with 1N HCl. The resultedprecipitate was collected by vacuum filtration, washed with methanol anddried to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.54 (s, 1H, NH), 11.15 (s, 1H, NH), 9.19(d, J=2 Hz, 1H), 8.88 (d, J=2 Hz, 1H), 8.3 (m, 1H), 7.72 (br t, J=5.8Hz, 1H), 7.26 (t, J=7.7 Hz, 1H), 7.01 (d, J=7.7 Hz, 1H), 6.89 (d, J=7.7Hz, 1H), 6.66 (s, 1H, H-vinyl), 3.52 (m, 2H), 3.18 (m, 6H), 2.43 (s, 3H,CH₃), 1.69 (s, 3H, CH₃), 1.21 (t, J=7.4 Hz, 6H, N(CH₂CH₃)₂).

MS +ve APCI m/z 502 [M⁺+1].

Example 735-[4-(2-Aminopyrimidin-5-yl)-2-oxo-1,2-dihydroindol-3-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylicAcid (2-diethylaminoethyl)amide

4-(2-Aminopyrimidin-5-yl)-1,3-dihydroindol-2-one (55 mg) was condensedwith 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (65 mg) to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.46 (s, 1H, NH), 11.02 (br s, 1H, NH),8.27 (s, 2H), 7.38 (t, 1H, NH), 7.17 (t, J=7.8 Hz, 1H), 7.02 (s, 1H,H-vinyl), 6.91 (d, J=7.8 Hz, 1H), 6.86 (s, 2H, NH₂), 6.79 (d, J=7.8 Hz,1H), 3.22 (m, 2H), 2.45-2.52 (m, 6H), 2.39 (s, 3H, CH₃), 1.90 (s, 3H,CH₃), 0.95 (t, J=7.0 Hz, 6H, N(CH₂CH₃)₂).

MS −ve APCI m/z 472 [M⁺−1].

Example 744-(2-Aminopyrimidin-5-yl)-3-[3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrol-2-ylmethylene]-1,3-dihydroindol-2-one

4-(2-Aminopyrimidin-5-yl)-1,3-dihydroindol-2-one (89 mg) was condensedwith3,5-dimethyl-4-(4-methylpiperazine-1-carbonyl)-1H-pyrrole-2-carbaldehyde(98 mg) to give the title compound.

¹H NMR (360 MHz, DMSO-d₆) δ 13.42 (s, 1H, NH), 11.01 (br s, 1H, NH),8.28 (s, 2H), 7.17 (t, J=7.7 Hz, 1H), 6.98 (s, 1H, H-vinyl), 6.91 (d,J=7.7 Hz, 1H), 6.86 (s, 2H, NH₂), 6.79 (d, J=7.7 Hz, 1H), 3.3-3.4 (br s,4H), 2.24 (br s, 4H), 2.16 (s, 3H, CH₃), 1.76 (s, 3H, CH₃).

MS −ve APCI m/z 456 [M⁺−1].

Example 752,4-Dimethyl-5-(2-oxo-4-pyridin-3-yl-1,2-dihydroindol-3-ylidenemethyl)-1H-pyrrole-3-carboxylicAcid (2-diethylaminoethyl) amide

A mixture of 4-pyridin-3-yl-1,3-dihydroindol-2-one (42 mg, 0.2 mmol),5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid(2-diethylaminoethyl)amide (53 mg, 0.2 mmol) and piperidine (0.1 mL) inethanol(1 mL) was heated in a sealed tube at 80° C. for 2 hours, heatingwas stopped and the tube was shaken at room temperature for 2 days. Asmall amount of water was added to the reaction and the solid whichformed was collected by vacuum filtration, washed with water and driedto give the title compound.

¹H NMR (400 MHz, DMSO-d₆) δ 13.47 (s, 1H, NH), 11.10 (s, 1H, NH), 8.71(dd, J=1.5 & 4.7 Hz, 1H), 8.64 (d, J=1.5 Hz, 1H), 7.89 (m, 1H), 7.58 (m,1H), 7.36 (t, J=5.5 Hz, 1H, NH), 7.23 (t, J=7.6 Hz, 1H, NH),6.98 (d,J=7.6 Hz, 1H), 6.83 (d, J=7.6 Hz, 1H), 6.68 (s, 1H, H-vinyl), 3.21 (m,2H), 2.5 (m, 6H), 2.39 (s, 3H, CH₃), 1.68 (s, 3H, CH₃), 0.95 (t, J=7.2Hz, 6H, N(CH₂CH₃)₂).

MS −ve APCI m/z 456 [M⁺−1].

Biological Evaluation

It will be appreciated that, in any given series of compounds, a rangeof biological activities will be observed. In its presently preferredembodiments, this invention relates to novel4-heteroaryl-3-heteroarylidenyl-2-indolinones demonstrating the abilityto modulate RTK, CTK, and STK activity.

The following assays are employed to explore the activity of thecompounds of this invention and to select those demonstrating thedesired level of activity against various target species.

A. Assay Procedures.

The following assays may be used to determine the level of activity andeffect of the different compounds of the present invention on one ormore PKs. Similar assays can be designed along the same lines for any PKusing techniques well known in the art.

Several of the assays described herein are performed in an ELISA(Enzyme-Linked Immunosorbent Sandwich Assay) format (Voller, et al.,1980, “Enzyme-Linked Immunosorbent Assay,” 1, Manual of ClinicalImmunology, 2 d ed., Rose and Friedman, Am. Soc. Of Microbiology,Washington, D.C., pp. 359-371). The general procedure is as follows: acompound is introduced to cells expressing the test kinase, eithernaturally or recombinantly, for a selected period of time after which,if the test kinase is a receptor, a ligand known to activate thereceptor is added. The cells are lysed and the lysate is transferred tothe wells of an ELISA plate previously coated with a specific antibodyrecognizing the substrate of the enzymatic phosphorylation reaction.Non-substrate components of the cell lysate are washed away and theamount of phosphorylation on the substrate is detected with an antibodyspecifically recognizing phosphotyrosine compared with control cellsthat were not contacted with a test compound.

The presently preferred protocols for conducting the ELISA experimentsfor specific PKs is provided below. However, adaptation of theseprotocols for determining the activity of compounds against other RTKs,as well as for CTKs and STKs, is well within the scope of knowledge ofthose skilled in the art. Other assays described herein measure theamount of DNA made in response to activation of a test kinase, which isa general measure of a proliferative response. The general procedure forthis assay is as follows: a compound is introduced to cells expressingthe test kinase, either naturally or recombinantly, for a selectedperiod of time after which, if the test kinase is a receptor, a ligandknown to activate the receptor is added. After incubation at leastovernight, a DNA labeling reagent such as 5-bromodeoxyuridine (BrdU) orH³-thymidine is added. The amount of labeled DNA is detected with eitheran anti-BrdU antibody or by measuring radioactivity and is compared tocontrol cells not contacted with a test compound.

GST-Flk-1 Bioassay

This assay analyzes the tyrosine kinase activity of GST-Flk1 onpoly(glu-tyr) peptides.

Materials and Reagents:

1. Corning 96-well ELISA plates (Corning Catalog No. 25805-96).

2. poly(glu-tyr) 4:1, lyophilizate (Sigma Catalog No. P0275), 1 mg/ml insterile PBS.

3. PBS Buffer: for 1 L, mix 0.2 g KH₂PO₄, 1.15 g Na₂HPO₄, 0.2 g KCl and8 g NaCl in approx. 900 ml dH₂O. When all reagents have dissolved,adjust the pH to 7.2 with HCl. Bring total volume to 1 L with dH₂O.

4. PBST Buffer: to 1 L of PBS Buffer, add 1.0 ml Tween-20.

5. TBB—Blocking Buffer: for 1 L, mix 1.21 g TRIS, 8.77 g NaCl, 1 mlTWEEN-20 in approximately 900 ml dH₂O. Adjust pH to 7.2 with HCl. Add 10g BSA, stir to dissolve. Bring total volume to 1 L with dH₂O. Filter toremove particulate matter.

6. 1% BSA in PBS: add 10 g BSA to approx. 990 ml PBS buffer, stir todissolve. Adjust total volume to 1 L with PBS buffer, filter to removeparticulate matter.

7. 50 mM Hepes pH 7.5.

8. GST-Flk1cd purified from sf9 recombinant baculovirus transformation(SUGEN, Inc.).

9. 4% DMSO in dH₂O.

10. 10 mM ATP in dH₂O.

11. 40 mM MnCl₂

12. Kinase Dilution Buffer (KDB): mix 10 ml Hepes (pH 7.5), 1 ml 5MNaCl, 40 μL 100 mM sodium orthovanadate and 0.4 ml of 5% BSA in dH₂Owith 88.56 ml dH₂O.

13. NUNC 96-well V bottom polypropylene plates, Applied ScientificCatalog # AS-72092

14. EDTA: mix 14.12 g ethylenediaminetetraacetic acid (EDTA) withapprox. 70 ml dH₂O. Add 10 N NaOH until EDTA dissolves. Adjust pH to8.0. Adjust total volume to 100 ml with dH₂O.

15. 10 and 20 Antibody Dilution Buffer: mix 10 ml of 5% BSA in PBSbuffer with 89.5 ml TBST.

16. Anti-phosphotyrosine rabbit polyclonal antisera (Sugen, Inc.)

17. Goat anti-rabbit HRP conjugate.

18. ABST solution: To approx. 900 ml dH₂O add 19.21 g citric acid and35.49 g Na₂HPO₄. Adjust pH to 4.0 with phosphoric acid. Add2,2′-Azinobis(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS, Sigma, Cat.No. A-1888, hold for approx. ½ hour, filter.

19. 30% Hydrogen Peroxide.

20. ABST/H₂O₂: add 3 μl of H₂O₂ to 15 ml of ABST solution.

21. 0.2 M HCl.

Procedure:

1. Coat Corning 96-well ELISA plates with 2 μg of polyEY in 100 μlPBS/well, hold at room temperature for 2 hours or at 4° C. overnight.Cover plates to prevent evaporation.

2. Remove unbound liquid from wells by inverting plate. Wash once withTBST. Pat the plate on a paper towel to remove excess liquid.

3. Add 100 μl of 1% BSA in PBS to each well. Incubate, with shaking, for1 hr. at room temperature.

4. Repeat step 2.

5. Soak wells with 50 mM HEPES (pH 7.5, 150 μl/well).

6. Dilute test compound with dH₂O/4% DMSO to 4 times the desired finalassay concentration in 96-well polypropylene plates.

7. Add 25 μl diluted test compound to each well of ELISA plate. Incontrol wells, place 25 μl of dH₂O/4% DMSO.

8. Dilute GST-Flk1 0.005 μg (5 ng)/well in KDB.

9. Add 50 μl of diluted enzyme to each well.

10. Add 25 μl 0.5 M EDTA to negative control wells.

11. Add 25 μl of 40 mM MnCl₂ with 4× ATP (2 μM) to all wells (100 μlfinal volume, 0.5 μM ATP final concentration in each well).

12. Incubate, with shaking, for 15 minutes at room temperature.

13. Stop reaction by adding 25 μl of 500 mM EDTA to each well.

14. Wash 3× with TBST and pat plate on paper towel to remove excessliquid.

15. Add 100 μl per well anti-phosphotyrosine antisera, 1:10,000 dilutionin antibody dilution buffer. Incubate, with shaking, for 90 min. at roomtemperature.

16. Wash as in step 14.

17. Add 100 μl/well of goat anti-rabbit HRP conjugate (1:6,000 inantibody dilution buffer). Incubate, with shaking, for 90 minutes areroom temperature.

18. Wash as in Step 14.

19. Add 100 μl room temperature ABST/H₂O₂ solution to each well.

20. Incubate, with shaking for 15 to 30 minutes at room temperature.

21. If necessary, stop reaction by adding 100 μl of 0.2 M HCl to eachwell.

22. Read results on Dynatech MR⁷⁰⁰⁰ ELISA reader with test filter at 410nM and reference filter at 630 nM.

PYK2 Bioassay

This assay is used to measure the in vitro kinase activity of HAepitope-tagged full length pyk2 (FL.pyk2-HA) in an ELISA assay.

Materials and Reagents:

1. Corning 96-well Elisa plates.

2. 12CA5 monoclonal anti-HA antibody (SUGEN, Inc.)

3. PBS (Dulbecco's Phosphate-Buffered Saline (Gibco Catalog #450-1300EB)

4. TBST Buffer: for 1 L, mix 8.766 g NaCl, 6.057 g TRIS and 1 ml of 0.1%Triton X-100 in approx. 900 ml dH₂O. Adjust pH to 7.2, bring volume to 1L.

5. Blocking Buffer: for 1 L, mix 100 g 10% BSA, 12.1 g 100 mM TRIS,58.44 g 1M NaCl and 10 mL of 1% TWEEN-20.

6. FL.pyk2-HA from sf9 cell lysates (SUGEN, Inc.).

7. 4% DMSO in MilliQue H₂O.

8. 10 mM ATP in dH₂O.

9. 1M MnCl₂.

10. 1M MgCl₂.

11. 1M Dithiothreitol (DTT).

12. 10× Kinase buffer phosphorylation: mix 5.0 ml 1M Hepes (pH 7.5), 0.2ml 1M MnCl₂, 1.0 ml 1 M MgCl₂, 1.0 ml 10% Triton X-100 in 2.8 ml dH₂O.Just prior to use, add 0.1 ml 1M DTT.

13. NUNC₉₆-well V bottom polypropylene plates.

14. 500 mM EDTA in dH₂O.

15. Antibody dilution buffer: for 100 mL, 1 mL 5% BSA/PBS and 1 mL 10%Tween-20 in 88 mL TBS.

16.HRP-conjugated anti-Ptyr (PY99, Santa Cruz Biotech Cat. No. SC-7020).

17. ABTS, Moss, Cat. No. ABST-2000.

18. 10% SDS.

Procedure:

1. Coat Corning 96 well ELISA plates with 0.5 μg per well 12CA5 anti-HAantibody in 100 μl PBS. Store overnight at 4° C.

2. Remove unbound HA antibody from wells by inverting plate. Wash platewith dH₂O. Pat the plate on a paper towel to remove excess liquid.

3. Add 150 μl Blocking Buffer to each well. Incubate, with shaking, for30 min at room temperature.

4. Wash plate 4× with TBS-T.

5. Dilute lysate in PBS (1.5 μg lysate/100 μl PBS).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 1 hr.

7. Wash as in step 4.

8. Add 50 μl of 2× kinase Buffer to ELISA plate containing capturedpyk2-HA.

9. Add 25 μL of 400 μM test compound in 4% DMSO to each well. Forcontrol wells use 4% DMSO alone.

10. Add 25 μL of 0.5 M EDTA to negative control wells.

11. Add 25 μl of 20 μM ATP to all wells. Incubate, with shaking, for 10minutes.

12. Stop reaction by adding 25 μl 500 mM EDTA (pH 8.0) to all wells.

13. Wash as in step 4.

14. Add 100 μL HRP conjugated anti-Ptyr diluted 1:6000 in AntibodyDilution Buffer to each well. Incubate, with shaking, for 1 hr. at roomtemperature.

15. Wash plate 3× with TBST and 1× with PBS.

16. Add 100 μL of ABST solution to each well.

17. If necessary, stop the development reaction by adding 20 μL 10% SDSto each well.

18. Read plate on ELISA reader with test filter at 410 nM and referencefilter at 630 nM.

FGFR1 Bioassay

This assay is used to measure the in vitro kinase activity of FGF1-R inan ELISA assay.

Materials and Reagents:

1. Costar 96-well Elisa plates (Corning Catalog #3369).

2. Poly(Glu-Tyr) (Sigma Catalog #P0275).

3. PBS (Gibco Catalog #450-1300EB)

4. 50 mM Hepes Buffer Solution.

5. Blocking Buffer (5% BSA/PBS).

6. Purified GST-FGFR1 (SUGEN, Inc.)

7. Kinase Dilution Buffer. Mix 500 μl 1M Hepes (GIBCO), 20 μl 5%BSA/PBS, 10 μl 100 mM sodium orthovanadate and 50 μl SM NaCl.

8. 10 mM ATP

9. ATP/MnCl₂ phosphorylation mix: mix 20 μL ATP, 400 μL 1M MnCl₂ and9.56 ml dH₂O.

10. NUNC₉₆-well V bottom polypropylene plates (Applied ScientificCatalog #AS-72092).

11. 0.5M EDTA.

12. 0.05% TBST Add 500 μL TWEEN to 1 liter TBS.

13. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

14. Goat anti-rabbit IgG peroxidase conjugate (Biosource, Catalog#ALI0404).

15. ABTS Solution.

16. ABTS/H₂O₂ solution.

Procedure:

1. Coat Costar 96 well ELISA plates with 1 μg per well Poly(Glu-Tyr) in100 μl PBS. Store overnight at 4° C.

2. Wash coated plates once with PBS.

3. Add 150 μL of 5%BSA/PBS Blocking Buffer to each well. Incubate, withshaking, for 1 hr at room temperature.

4. Wash plate 2× with PBS, then once with 50 mM Hepes. Pat plates on apaper towel to remove excess liquid and bubbles.

5. Add 25μL of 0.4 mM test compound in 4% DMSO or 4% DMSO alone(controls) to plate.

6. Dilute purified GST-FGFR1 in Kinase Dilution Buffer (5 ng kinase/50ul KDB/well).

7. Add 50 μL of diluted kinase to each well.

8. Start kinase reaction by adding 25 μl/well of freshly preparedATP/Mn++ (0.4 ml 1M MnCl₂, 40 μL 10 mM ATP, 9.56 ml dH₂O), freshlyprepared).

9. Stop reaction with 25μL of 0.5M EDTA.

10. Wash plate 4× with fresh TBST.

11. Make up Antibody Dilution Buffer: For 50 ml, mix 5 ml of 5% BSA, 250μl of 5% milk and 50 μl of 100 mM sodium vanadate, bring to final volumewith 0.05% TBST.

12. Add 100 μl per well of anti-phosphotyrosine (1:10000 dilution inADB). Incubate, with shaking for 1 hr. at room temperature.

13. Wash as in step 10.

14. Add 100 μl per well of Biosource Goat anti-rabbit IgG peroxidaseconjugate (1:6000 dilution in ADB). Incubate, with shaking for 1 hr. atroom temperature.

15. Wash as in step 10 and then with PBS to remove bubbles and excessTWEEN.

16. Add 100 μl of ABTS/H₂O₂ solution to each well.

17. Incubate, with shaking, for 10 to 20 minutes. Remove any bubbles.

18. Read assay on Dynatech MR⁷⁰⁰⁰ elisa reader: test filter at 410 nM,reference filter at 630 nM.

EGFR Bioassay

This assay is used to the in vitro kinase activity of EGFR in an ELISAassay.

Materials and Reagents:

1. Corning 96-well Elisa plates.

2. SUMO1 monoclonal anti-EGFR antibody (SUGEN, Inc.).

3. PBS.

4. TBST Buffer.

5. Blocking Buffer: for 100 ml, mix 5.0 g Carnation® Instant Non-fatMilk with 100 ml of PBS.

6. A431 cell lysate (SUGEN, Inc.).

7. TBS Buffer.

8. TBS +10% DMSO: for 1L, mix 1.514 g TRIS, 2.192 g NaCl and 25 ml DMSO;bring to 1 liter total volume with dH₂O.

9. ATP (Adenosine-5′-triphosphate, from Equine muscle, Sigma Cat. No.A-5394), 1.0 mM solution in dH₂O. This reagent should be made upimmediately prior to use and kept on ice.

10. 1.0 mM MnCl₂.

11. ATP/MnCl₂ phosphorylation mix: for 10 ml, mix 300 μl of 1 mM ATP,500 μl MnCl₂ and 9.2 ml dH₂O. Prepare just prior to use, keep on ice.

12. NUNC₉₆-well V bottom polypropylene plates.

13. EDTA.

14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALI0404).

16. ABTS.

17. 30% Hydrogen peroxide.

18. ABTS/H₂O₂.

19. 0.2 M HCl.

Procedure:

1. Coat Corning 96 well ELISA plates with 0.5 μg SUMO1 in 100 μl PBS perwell, hold overnight at 4° C.

2. Remove unbound SUMO1 from wells by inverting plate to remove liquid.Wash 1× with dH₂O. Pat the plate on a paper towel to remove excessliquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate, with shaking,for 30 min. at room temperature.

4. Wash plate 3× with deionized water, then once with TBST. Pat plate ona paper towel to remove excess liquid and bubbles.

5. Dilute lysate in PBS (7 μg lysate/100 μl PBS).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 1 hr.

7. Wash plates as in 4, above.

8. Add 120 μl TBS to ELISA plate containing captured EGFR.

9. Dilute test compound 1:10 in TBS, place in well

10. Add 13.5 μl diluted test compound to ELISA plate. To control wells,add 13.5 μl TBS in 10% DMSO.

11. Incubate, with shaking, for 30 minutes at room temperature.

12. Add 15 μl phosphorylation mix to all wells except negative controlwell. Final well volume should be approximately 150 μl with 3 μM ATP/5mM MnCl₂ final concentration in each well. Incubate with shaking for 5minutes.

13. Stop reaction by adding 16.5 μl of EDTA solution while shaking.Shake for additional 1 min.

14. Wash 4× with deionized water, 2× with TBST.

15. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) per well.Incubate, with shaking, for 30-45 min. at room temperature.

16. Wash as in 4, above.

17. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate(1:2000 dilution in TBST) to each well. Incubate with shaking for 30min. at room temperature.

18. Wash as in 4, above.

19. Add 100 μl of ABTS/H₂O₂ solution to each well.

20. Incubate 5 to 10 minutes with shaking. Remove any bubbles.

21. If necessary, stop reaction by adding 100 μl 0.2 M HCl per well.

22. Read assay on Dynatech MR⁷⁰⁰⁰ ELISA reader: test filter at 410 nM,reference filter at 630 nM.

PDGFR Bioassay

This assay is used to the in vitro kinase activity of PDGFR in an ELISAassay.

Materials and Reagents:

1. Corning 96-well Elisa plates

2. 28D4C10 monoclonal anti-PDGFR antibody (SUGEN, Inc.).

3. PBS.

4. TBST Buffer.

5. Blocking Buffer (same as for EGFR bioassay).

6. PDGFR-β expressing NIH 3T3 cell lysate (SUGEN, Inc.).

7. TBS Buffer.

8. TBS +10% DMSO.

9. ATP.

10. MnCl₂.

11. Kinase buffer phosphorylation mix: for 10 ml, mix 250 μl 1M TRIS,200 μl 5M NaCl, 100 μl 1M MnCl₂ and 50 μl 100 mM Triton X-100 in enoughdH₂O to make 10 ml.

12. NUNC₉₆-well V bottom polypropylene plates.

13. EDTA.

14. Rabbit polyclonal anti-phosphotyrosine serum (SUGEN, Inc.).

15. Goat anti-rabbit IgG peroxidase conjugate (Biosource Cat. No.ALI0404).

16. ABTS.

17.Hydrogen peroxide, 30% solution.

18. ABTS/H₂O₂.

19. 0.2 M HCl.

Procedure:

1. Coat Corning 96 well ELISA plates with 0.5 μg 28D4C10 in 100 μl PBSper well, hold overnight at 4° C.

2. Remove unbound 28D4C10 from wells by inverting plate to removeliquid. Wash 1× with dH₂O. Pat the plate on a paper towel to removeexcess liquid.

3. Add 150 μl of Blocking Buffer to each well. Incubate for 30 min. atroom temperature with shaking.

4. Wash plate 3× with deionized water, then once with TBST. Pat plate ona paper towel to remove excess liquid and bubbles.

5. Dilute lysate in HNTG (10 μg lysate/100 μl HNTG).

6. Add 100 μl of diluted lysate to each well. Shake at room temperaturefor 60 min.

7. Wash plates as described in Step 4.

8. Add 80 μl working kinase buffer mix to ELISA plate containingcaptured PDGFR.

9. Dilute test compound 1:10 in TBS in 96-well polypropylene plates.

10. Add 10 μl diluted test compound to ELISA plate. To control wells,add 10 μl TBS +10% DMSO. Incubate with shaking for 30 minutes at roomtemperature.

11. Add 10 μl ATP directly to all wells except negative control well(final well volume should be approximately 100 μl with 20 μM ATP in eachwell.) Incubate 30 minutes with shaking.

12. Stop reaction by adding 10 μl of EDTA solution to each well.

13. Wash 4× with deionized water, twice with TBST.

14. Add 100 μl anti-phosphotyrosine (1:3000 dilution in TBST) per well.Incubate with shaking for 30-45 min. at room temperature.

15. Wash as in Step 4.

16. Add 100 μl Biosource Goat anti-rabbit IgG peroxidase conjugate(1:2000 dilution in TBST) to each well. Incubate with shaking for 30min. at room temperature.

17. Wash as in Step 4.

18. Add 100 μl of ABTS/H₂O₂ solution to each well.

19. Incubate 10 to 30 minutes with shaking. Remove any bubbles.

20. If necessary stop reaction with the addition of 100 μl 0.2 M HCl perwell.

21. Read assay on Dynatech MR⁷⁰⁰⁰ ELISA reader with test filter at 410nM and reference filter at 630 nM.

Cellular HER-2 Kinase Assay

This assay is used to measure HER-2 kinase activity in whole cells in anELISA format.

Materials and Reagents:

1. DMEM (GIBCO Catalog #11965-092).

2. Fetal Bovine Serum (FBS, GIBCO Catalog #16000-044), heat inactivatedin a water bath for 30 min. at 56° C.

3. Trypsin (GIBCO Catalog #25200-056).

4. L-Glutamine (GIBCO Catalog #25030-081).

5.HEPES (GIBCO Catalog #15630-080).

6. Growth Media: Mix 500 ml DMEM, 55 ml heat inactivated FBS, 10 mlHEPES and 5.5 ml L-Glutamine.

7. Starve Media: Mix 500 ml DMEM, 2.5 ml heat inactivated FBS, 10 mlHEPES and 5.5 ml L-Glutamine. 8. PBS.

9. Flat Bottom 96-well Tissue Culture Micro Titer Plates (CorningCatalog #25860).

10. 15 cm Tissue Culture Dishes (Corning Catalog #08757148).

11. Corning 96-well ELISA Plates.

12. NUNC₉₆-well V bottom polypropylene plates.

13. Costar Transfer Cartridges for the Transtar 96 (Costar Catalog#7610).

14. SUMO 1: monoclonal anti-EGFR antibody (SUGEN, Inc.).

15. TBST Buffer.

16. Blocking Buffer: 5% Carnation Instant Milk® in PBS.

17. EGF Ligand: EGF-201, Shinko American, Japan. Suspend powder in 100uL of 10 mM HCl. Add 100 uL 10 mM NaOH. Add 800 uL PBS and transfer toan Eppendorf tube, store at −20° C. until ready to use.

18.HNTG Lysis Buffer: For Stock 5× HNTG, mix 23.83 g Hepes, 43.83 gNaCl, 500 ml glycerol and 100 ml Triton X-100 and enough dH₂O to make 1L of total solution.

For 1× HNTG*, mix 2 ml 5× HNTG, 100 μL 0.1M Na₃VO₄, 250 μL 0.2M Na₄P₂O₇and 100 μL EDTA.

19. EDTA.

20. Na₃VO₄: To make stock solution, mix 1.84 g Na₃VO₄ with 90 ml dH₂O.Adjust pH to 10. Boil in microwave for one minute (solution becomesclear). Cool to room temperature. Adjust pH to 10. Repeatheating/cooling cycle until pH remains at 10.

21. 200 mM Na₄P₂O₇.

22. Rabbit polyclonal antiserum specific for phosphotyrosine (anti-Ptyrantibody, SUGEN, Inc.).

23. Affinity purified antiserum, goat anti-rabbit IgG antibody,peroxidase conjugate (Biosource Cat #ALI0404).

24. ABTS Solution.

25. 30% Hydrogen peroxide solution.

26. ABTS/H₂O₂.

27. 0.2 M HCl.

Procedure:

1. Coat Corning 96 well ELISA plates with SUMO1 at 1.0 ug per well inPBS, 100 ul final volume/well. Store overnight at 4° C.

2. On day of use, remove coating buffer and wash plate

3 times with dH₂O and once with TBST buffer. All washes in this assayshould be done in this manner, unless otherwise specified.

3. Add 100 ul of Blocking Buffer to each well. Incubate plate, withshaking, for 30 min. at room temperature. Just prior to use, wash plate.

4. Use EGFr/HER-2 chimera/3T3-C7 cell line for this assay.

5. Choose dishes having 80-90% confluence. Collect cells bytrypsinization and centrifuge at 1000 rpm at room temperature for 5 min.

6. Resuspend cells in starve medium and count with trypan blue.Viability above 90% is required. Seed cells in starve medium at adensity of 2,500 cells per well, 90 ul per well, in a 96 well microtiterplate. Incubate seeded cells overnight at 37° under 5% CO₂.

7. Start the assay two days after seeding.

8. Test compounds are dissolved in 4% DMSO. Samples are then furtherdiluted directly on plates with starve-DMEM. Typically, this dilutionwill be 1:10 or greater. All wells are then transferred to the cellplate at a further 1:10 dilution (10μl sample and media into 90 μl ofstarve media). The final DMSO concentration should be 1% or lower. Astandard serial dilution may also be used.

9. Incubate under 5% CO₂ at 37° C. for 2 hours.

10. Prepare EGF ligand by diluting stock EGF (16.5 uM) in warm DMEM to150 nM.

11. Prepare fresh HNTG* sufficient for 100 ul per well; place on ice.

12. After 2 hour incubation with test compound, add prepared EGF ligandto cells, 50 ul per well, for a final concentration of 50 nM. Positivecontrol wells receive the same amount of EGF. Negative controls do notreceive EGF. Incubate at 37° C. for 10 min.

13. Remove test compound, EGF, and DMEM. Wash cells once with PBS.

14. Transfer HNTG* to cells, 100 ul per well. Place on ice for 5minutes. Meanwhile, remove blocking buffer from ELISA plate and wash.

15. Scrape cells from plate with a micropipettor and homogenize cellmaterial by repeatedly aspirating and dispensing the HNTG* lysis buffer.Transfer lysate to a coated, blocked, washed ELISA plate.

16. Incubate, with shaking, at room temperature for 1 hr.

17. Remove lysate, wash. Transfer freshly diluted anti-Ptyr antibody(1:3000 in TBST) to ELISA plate, 100 ul per well.

18. Incubate, with shaking, at room temperature, for 30 min.

19. Remove anti-Ptyr antibody, wash. Transfer freshly diluted BIOSOURCEantibody to ELISA plate(1:8000 in TBST, 100 ul per well).

20. Incubate, with shaking, at room temperature for 30 min.

21. Remove BIOSOURCE antibody, wash. Transfer freshly prepared ABTS/H₂O₂solution to ELISA plate, 100 ul per well.

22. Incubate, with shaking, for 5-10 minutes. Remove any bubbles.

23. Stop reaction by adding 100 ul of 0.2M HCl per well.

24. Read assay on Dynatech MR⁷⁰⁰⁰ ELISA reader with test filter set at410 nM and reference filter at 630 nM.

Cdk2/Cyclin A Assay

This assay is used to measure the in vitro serine/threonine kinaseactivity of human cdk2/cyclin A in a Scintillation Proximity Assay(SPA).

Materials and Reagents.

1. Wallac 96-well polyethylene terephthalate (flexi) plates (WallacCatalog # 1450-401).

2. Amersham Redivue [γ³³P] ATP (Amersham catalog #AH 9968).

3. Amersham streptavidin coated polyvinyltoluene SPA beads (Amershamcatalog #RPNQ0007). The beads should be reconstituted in PBS withoutmagnesium or calcium, at 20 mg/ml.

4. Activated cdk2/cyclin A enzyme complex purified from Sf9 cells(SUGEN, Inc.).

5. Biotinylated peptide substrate (Debtide). Peptide biotin-X-PKTPKKAKKLis dissolved in dH₂O at a concentration of 5 mg/ml.

6. 20% DMSO in dH₂O.

7. Kinase buffer: for 10 ml, mix 9.1 ml dH₂O, 0.5 ml TRIS(pH 7.4), 0.2ml 1M MgCl₂, 0.2 ml 10% NP40 and 0.02 ml 1M DTT, added fresh just priorto use.

8. 10 mM ATP in dH₂O.

9. 1M Tris, pH adjusted to 7.4 with HCl.

10. 1M MgCl₂.

11. 1M DTT.

12. PBS (Gibco Catalog #14190-144).

13. 0.5M EDTA.

14. Stop solution: For 10 ml, mix 9.25 ml PBS, 0.05 ml

15. 10 mM ATP, 0.1 ml 0.5 M EDTA, 0.1 ml 10% Triton X-100 and 1.5 ml of50 mg/ml SPA beads.

Procedure:

1. Prepare solutions of test compounds at 4× the desired finalconcentration in 5% DMSO. Add 10 ul to each well. For positive andnegative controls, use 10 ul 20% DMSO alone in wells.

2. Dilute the peptide substrate (deb-tide) 1:250 with dH₂O to give afinal concentration of 0.02 mg/ml.

3. Mix 24 ul 0.1 mM ATP with 24 uCi γ³³P ATP and enough dH₂O to make 600ul.

4. Mix diluted peptide and ATP solutions 1:1 (600 ul+600 ul per plate).Add 10 ul of this solution to each well.

5. Dilute 5 ul of cdk2/cyclin A solution into 2.1 ml 2× kinase buffer(per plate). Add 20 ul enzyme per well. For negative controls, add 20 ul2× kinase buffer without enzyme.

6. Mix briefly on a plate shaker; incubate for 60 minutes.

7. Add 200 ul stop solution per well.

8. Let stand at least 10 min.

9. Spin plate at approx. 2300 rpm for 10-15 min.

10. Count plate on Trilux reader.

Met Transphosphorylation Assay

This assay is used to measure phosphotyrosine levels on a poly(glutamicacid:tyrosine, 4:1) substrate as a means for identifyingagonists/antagonists of met transphosphorylation of the substrate.

Materials and Reagents:

1. Corning 96-well Elisa plates, Corning Catalog #25805-96.

2. Poly(glu-tyr), 4:1, Sigma, Cat. No; P 0275.

3. PBS, Gibco Catalog # 450-1300EB

4. 50 mM HEPES

5. Blocking Buffer: Dissolve 25 g Bovine Serum Albumin, Sigma Cat. NoA-7888, in 500 ml PBS, filter through a 4 μm filter.

6. Purified GST fusion protein containing the Met kinase domain, Sugen,Inc.

7. TBST Buffer.

8. 10% aqueous (MilliQue H₂O) DMSO.

9. 10 mM aqueous (dH₂O) Adenosine-5′-triphosphate, Sigma Cat. No.A-5394.

10. 2× Kinase Dilution Buffer: for 100 ml, mix 10 mL 1M HEPES at pH 7.5with 0.4 mL 5% BSA/PBS, 0.2 mL 0.1 M sodium orthovanadate and 1 mL 5Msodium chloride in 88.4 mL dH₂O.

11. 4× ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M manganesechloride and 0.02 mL 0.1 M ATP in 9.56 mL dH₂O.

12. 4× Negative Controls Mixture: for 10 mL, mix 0.4 mL 1 M manganesechloride in 9.6 mL dH₂O.

13. NUNC₉₆-well V bottom polypropylene plates, Applied ScientificCatalog #S-72092

14. 500 mM EDTA.

15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA/PBS, 0.5 mL5% Carnation® Instant Milk in PBS and 0.1 mL 0.1 M sodium orthovanadatein 88.4 mL TBST.

16. Rabbit polyclonal antophosphotyrosine antibody, Sugen, Inc.

17. Goat anti-rabbit horseradish peroxidase conjugated antibody,Biosource, Inc.

18. ABTS Solution: for 1 L, mix 19.21 g citric acid, 35.49 g Na₂HPO₄ and500 mg ABTS with sufficient dH₂O to make 1 L.

19. ABTS/H₂O₂: mix 15 mL ABST solution with 2 μL H₂O₂ five minutesbefore use.

20. 0.2 M HCl

Procedure:

1. Coat ELISA plates with 2 μg Poly(Glu-Tyr) in 100 μL PBS, holdovernight at 4° C.

2. Block plate with 150 μL of 5% BSA/PBS for 60 min.

3. Wash plate twice with PBS then once with 50 mM Hepes buffer pH 7.4.

4. Add 50 μl of the diluted kinase to all wells. (Purified kinase isdiluted with Kinase Dilution Buffer. Final concentration should be 10ng/well.)

5. Add 25 μL of the test compound (in 4% DMSO) or DMSO alone (4% indH₂O) for controls to plate.

6. Incubate the kinase/compound mixture for 15 minutes.

7. Add 25 μL of 40 mM MnCl₂ to the negative control wells.

8. Add 25 μL ATP/MnCl₂ mixture to the all other wells (except thenegative controls). Incubate for 5 min.

9. Add 25 μL 500 mM EDTA to stop reaction.

10. Wash plate 3× with TBST.

11. Add 100 μL rabbit polyclonal anti-Ptyr diluted 1:10,000 in AntibodyDilution Buffer to each well. Incubate, with shaking, at roomtemperature for one hour.

12. Wash plate 3× with TBST.

13. Dilute Biosource HRP conjugated anti-rabbit antibody 1:6,000 inAntibody Dilution buffer. Add 100 μL per well and incubate at roomtemperature, with shaking, for one hour.

14. Wash plate 1× with PBS.

15. Add 100 μl of ABTS/H₂O₂ solution to each well.

16. If necessary, stop the development reaction with the addition of 100μl of 0.2M HCl per well.

17. Read plate on Dynatech MR⁷⁰⁰⁰ elisa reader with the test filter at410 nM and the reference filter at 630 nM.

IGF-1 Transphosphorylation Assay

This assay is used to measure the phosphtyrosine level in poly(glutamicacid:tyrosine, 4:1) for the identification of agonists/antagonists ofgst-IGF-1 transphosphorylation of a substrate.

Materials and Reagents:

1. Corning 96-well Elisa plates.

2. Poly(Glu-tyr),4:1, Sigma Cat. No. P 0275.

3. PBS, Gibco Catalog #450-1300EB.

4. 50 mM HEPES

5. TBB Blocking Buffer: for 1 L, mix 100 g BSA, 12.1 gTRIS (pH 7.5),58.44 g sodium chloride and 10 mL 1%TWEEN-20.

6. Purified GST fusion protein containing the IGF-1 kinase domain(Sugen, Inc.)

7. TBST Buffer: for 1 L, mix 6.057 g Tris, 8.766 g sodium chloride and0.5 ml TWEEN-20 with enough dH₂O to make 1 liter.

8. 4% DMSO in Milli-Q H₂O.

9. 10 mM ATP in dH₂O.

10. 2× Kinase Dilution Buffer: for 100 mL, mix 10 mL 1 M HEPES (pH 7.5),0.4 mL 5% BSA in dH₂O, 0.2 mL 0.1 M sodium orthovanadate and 1 mL 5 Msodium chloride with enough dH₂O to make 100 mL.

11. 4× ATP Reaction Mixture: for 10 mL, mix 0.4 mL 1 M MnCl₂ and 0.008mL 0.01 M ATP and 9.56 mL dH₂O.

12. 4× Negative Controls Mixture: mix 0.4 mL 1 M MnCl₂ in 9.60 mL dH₂O.

13. NUNC₉₆-well V bottom polypropylene plates.

14. 500 mM EDTA in dH₂O.

15. Antibody Dilution Buffer: for 100 mL, mix 10 mL 5% BSA in PBS, 0.5mL 5% Carnation Instant Non-fat Milk in PBS and 0.1 mL 0.1 M sodiumorthovanadate in 88.4 mL TBST.

16. Rabbit Polyclonal antiphosphotyrosine antibody, Sugen, Inc.

17. Goat anti-rabbit HRP conjugated antibody, Biosource.

18. ABTS Solution.

20. ABTS/H₂O₂: mix 15 mL ABTS with 2 μL H₂O₂ 5 minutes before using.

21. 0.2 M HCl in dH₂O.

Procedure:

1. Coat ELISA plate with 2.0 μg/well Poly(Glu, Tyr), 4:1 (Sigma P0275)in 100 μl PBS. Store plate overnight at 4° C.

2. Wash plate once with PBS.

3. Add 100 μl of TBB Blocking Buffer to each well. Incubate plate for 1hour with shaking at room temperature.

4. Wash plate once with PBS, then twice with 50 mM Hepes buffer pH 7.5.

5. Add 25 μL of test compound in 4% DMSO (obtained by diluting a stocksolution of 10 mM test compound in 100% DMSO with dH₂O) to plate.

6. Add 10.0 ng of gst-IGF-1 kinase in 50 μl Kinase Dilution Buffer toall wells.

7. Start kinase reaction by adding 25 μl 4× ATP Reaction Mixture to alltest wells and positive control wells. Add 25 μl 4× Negative ControlsMixture to all negative control wells. Incubates for 10 minutes, withshaking, at room temperature.

8. Add 25 μl 0.5M EDTA (pH 8.0) to all wells.

9. Wash plate 4× with TBST Buffer.

10. Add rabbit polyclonal anti-phosphotyrosine antisera at a dilution of1:10,000 in 100 μl Antibody Dilution Buffer to all wells. Incubate, withshaking, at room temperature for 1 hour.

11. Wash plate as in step 9.

12. Add 100 μL Biosource anti-rabbit HRP at a dilution of 1:10,000 inAntibody dilution buffer to all wells. Incubate, with shaking, at roomtemperature for 1 hour.

13. Wash plate as in step 9, follow with one wash with PBS to removebubbles and excess Tween-20.

14. Develop by adding 100 μl/well ABTS/H₂O₂ to each well.

15. After about 5 minutes, read on ELISA reader with test filter at 410nm and referenced filter at 630 nm.

BrdU Incorporation Assays

The following assays use cells engineered to express a selected receptorand then evaluate the effect of a compound of interest on the activityof ligand-induced DNA synthesis by determining BrdU incorporation intothe DNA.

The following materials, reagents and procedure are general to each ofthe following BrdU incorporation assays. Variances in specific assaysare noted.

General Materials and Reagents:

1. The appropriate ligand.

2. The appropriate engineered cells.

3. BrdU Labeling Reagent: 10 mM, in PBS, pH7.4(Roche MolecularBiochemicals, Indianapolis, Ind.).

4. FixDenat: fixation solution (Roche Molecular Biochemicals,Indianapolis, Ind.).

5. Anti-BrdU-POD: mouse monoclonal antibody conjugated with peroxidase(Chemicon, Temecula, Calif.).

6. TMB Substrate Solution: tetramethylbenzidine (TMB, ready to use,Roche Molecular Biochemicals, Indianapolis, Ind.).

7. PBS Washing Solution: 1× PBS, pH 7.4.

8. Albumin, Bovine (BSA), fraction V powder (Sigma

General Procedure:

1. Cells are seeded at 8000 cells/well in 10% CS, 2 mM Gln in DMEM, in a96 well plate. Cells are incubated overnight at 37° C. in 5% CO₂.

2. After 24 hours, the cells are washed with PBS, and then areserum-starved in serum free medium (0%CS DMEM with 0.1% BSA) for 24hours.

3. On day 3, the appropriate ligand and the test compound are added tothe cells simultaneously. The negative control wells receive serum freeDMEM with 0.1% BSA only; the positive control cells receive the ligandbut no test compound. Test compounds are prepared in serum free DMEMwith ligand in a 96 well plate, and serially diluted for 7 testconcentrations.

4. After 18 hours of ligand activation, diluted BrdU labeling reagent(1:100 in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU(final concentration is 10 μM) for 1.5 hours.

5. After incubation with labeling reagent, the medium is removed bydecanting and tapping the inverted plate on a paper towel. FixDenatsolution is added (50 μl/well) and the plates are incubated at roomtemperature for 45 minutes on a plate shaker.

6. The FixDenat solution is removed by decanting and tapping theinverted plate on a paper towel. Milk is added (5% dehydrated milk inPBS, 200 μl/well) as a blocking solution and the plate is incubated for30 minutes at room temperature on a plate shaker.

7. The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution is added (1:200 dilution inPBS, 1% BSA, 50 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

8. The antibody conjugate is removed by decanting and rinsing the wells5 times with PBS, and the plate is dried by inverting and tapping on apaper towel.

9. TMB substrate solution is added (100 μl/well) and incubated for 20minutes at room temperature on a plate shaker until color development issufficient for photometric detection.

10. The absorbance of the samples are measured at 410 nm (in “dualwavelength” mode with a filter reading at 490 nm, as a referencewavelength) on a Dynatech ELISA plate reader.

EGF-Induced BrdU Incorporation Assay

Materials and Reagents:

1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

2. 3T3/EGFRc7.

Remaining Materials and Reagents and Procedure, as above.

EGF-Induced Her-2-Driven BrdU Incorporation Assay

Materials and Reagents:

1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

2. 3T3/EGFr/Her2/EGFr (EGFr with a Her-2 kinase domain).

Remaining Materials and Reagents and Procedure, as above.

EGF-Induced Her-4-Driven BrdU Incorporation Assay

Materials and Reagents:

1. Mouse EGF, 201 (Toyobo Co., Ltd., Japan).

2. 3T3/EGFr/Her4/EGFr (EGFr with a Her-4 kinase domain).

Remaining Materials and Reagents and Procedure, as above.

PDGF-Induced BrdU Incorporation Assay

Materials and Reagents:

1.Human PDGF B/B (Boehringer Mannheim, Germany).

2. 3T3/EGFRc7.

Remaining Materials and Reagents and Procedure, as above.

FGF-Induced BrdU Incorporation Assay

Materials and Reagents:

1.Human FGF2/bFGF (Gibco BRL, USA).

2. 3T3c7/EGFr

Remaining Materials and Reagents and Procedure, as above.

IGF1-Induced BrdU Incorporation Assay

Materials and Reagents:

1.Human, recombinant (G511, Promega Corp., USA)

2. 3T3/IGF1r.

Remaining Materials and Reagents and Procedure, as above.

Insulin-Induced BrdU Incorporation Assay

Materials and Reagents:

1. Insulin, crystalline, bovine, Zinc (13007, Gibco BRL, USA).

2. 3T3/H25.

Remaining Materials and Reagents and Procedure, as above.

HGF-Induced BrdU Incorporation Assay

Materials and Reagents:

1. Recombinant human HGF (Cat. No. 249-HG, R&D Systems, Inc. USA).

2. BxPC-3 cells (ATCC CRL-1687).

Remaining Materials and Reagents, as above.

Procedure:

1. Cells are seeded at 9000 cells/well in RPMI 10% FBS in a 96 wellplate. Cells are incubated overnight at 37 C in 5% CO₂.

2. After 24 hours, the cells are washed with PBS, and then are serumstarved in 100 μl serum-free medium (RPMI with 0.1% BSA) for 24 hours.

3. On day 3, 25 μl containing ligand (prepared at 1 μg/ml in RPMI with0.1% BSA; final HGF conc. is 200 ng/ml) and test compounds are added tothe cells. The negative control wells receive 25 μl serum-free RPMI with0.1% BSA only; the positive control cells receive the ligand (HGF) butno test compound. Test compounds are prepared at 5 times their finalconcentration in serum-free RPMI with ligand in a 96 well plate, andserially diluted to give 7 test concentrations. Typically, the highestfinal concentration of test compound is 100 μM, and 1:3 dilutions areused (i.e. final test compound concentration range is 0.137-100 μM).

4. After 18 hours of ligand activation, 12.5 μl of diluted BrdU labelingreagent (1:100 in RPMI, 0.1% BSA) is added to each well and the cellsare incubated with BrdU (final concentration is 10 μM) for 1 hour.

5. Same as General Procedure.

6. Same as General Procedure.

7. The blocking solution is removed by decanting and the wells arewashed once with PBS. Anti-BrdU-POD solution (1:100 dilution in PBS, 1%BSA) is added (100 μl/well) and the plate is incubated for 90 minutes atroom temperature on a plate shaker.

8. Same as General Procedure.

9. Same as General Procedure.

10. Same as General Procedure.

Exponential BrdU Incorporation Assay

This assay is used to measure the proliferation (DNA synthesis) ofexponentially growing A431 cells. The assay will screen for compoundsthat inhibit cell cycle progression.

Materials and Reagents:

Healthy growing A431 cells. The remainder of the Materials and Reagentsare the same as listed above in the general protocol section.

Procedure:

1. A431 cells are seeded at 8000 cells/well in 10% FBS, 2 mM Gln inDMEM, on a 96-well plate. Cells are incubated overnight at 37° C. in 5%CO₂.

2. On day 2, test compounds are serially diluted to 7 testconcentrations in the same growth medium on a 96-well plate and then areadded to the cells on a 96-well tissue culture plate.

3. After 20-24 hours of incubation, diluted BrdU labeling reagent (1:100in DMEM, 0.1% BSA) is added and the cells are incubated with BrdU (finalconcentration is 10 μM) for 2 hours.

Steps 5-10 of the General Procedure are used to complete the assay.

ZenSrc Assay

This assay is used to screen for inhibitors of the tyrosine kinase Src.

Materials and Reagents:

1. Coating buffer: PBS containing sodium azide (0.2 mg/ml).

2. 1% w/v BSA in PBS.

3. Wash buffer: PBS containing 0.05% v/v Tween 20 (PBS-TWEEN)

4. 500 mM HEPES pH 7.4.

5. ATP (40 μM)+MgCl₂ (80 mM) in distilled water.

6. MgCl₂ (80 mM) in distilled water (for no ATP blanks)

7. Test compounds, 10 mM in DMSO.

8. Assay Buffer: 100 mM HEPES, pH 7.4, containing 2 mM DTT, 0.2 mMsodium orthovanadate and 0.2 mgs/ml BSA.

9. Partially purified recombinant human Src (UBI (14-117)

10. Anti-phosphotyrosine (SUGEN rabbit polyclonal anti-PY)

11.HRP-linked goat anti-rabbit Ig (Biosource International #6430)

12.HRP substrate ABTS or Pierce Peroxidase substrate.

13. Corning ELISA plates.

Procedure:

1. Coat plates with 100 μl of 20 μg/ml poly(Glu-Tyr) (Sigma Cat.No.P0275) containing 0.01% sodium azide. Hold overnight at 4° C.

2. Block with 1% BSA at 100 μl/well for one hour at room temperature.

3. Plate test compounds (10 mM in DMSO) at 2 ul/well on a Costar plateready for dilution with dH₂O and plating to reaction plates.

4. Dilute Src kinase 1:10,000 in Reaction Buffer, for 5 plates prepare25 ml as follows: 2.5 mls 1M HEPES pH 7.4 (stored sterile at 4° C.),21.85 ml distilled water, 0.1 ml 5% BSA, 0.5 ml 10 mM sodiumorthovanadate (stored sterile at 4° C.), 50 μl 1.0M DTT (stored frozenat −20° C.), and 2.5 μl Src Kinase (stored frozen at −80° C.).

5. Add 48 μl of distilled water to the 2 μl of each compound in thedilution plate then add 25 μl/well of this to the reaction plate.

6. Add 50 μl of HRP to each reaction buffer well and then 25 μlATP-MgCl₂/well (MgCl₂ only to no ATP blanks) Incubate at roomtemperature for 15 minutes on plate shaker. Stop reaction by adding 25μl of 0.5M EDTA to each well.

7. Wash 4× with PBS-TWEEN.

8. Add 100 μl anti-phosphotyrosine (1:10,000 of anti-pTyr serum or1:3,000 of 10% glycerol diluted PA-affinity purified antibody) inPBS-TWEEN containing 0.5% BSA, 0.025% Non-fat milk powder and 100 uMsodium orthovanadate. Incubate with continuous shaking at roomtemperature for one hour.

9. Wash plates 4× with PBS-TWEEN.

10. Add 100 μl HRP-linked Ig (1:5,000) in PBS-TWEEN containing 0.5% BSA,0.025% Non-fat milk powder, 100 μM sodium orthovanadate. Incubate withshaking at room temperature for one hour.

11. Wash plates 4× with PBS-TWEEN and then once with PBS.

12. Develop plate using ABTS or other peroxidase substrate.

Cell Cycle Assay:

A431 cells in standard growth medium are exposed to a desiredconcentration of a test compound for 20-24 hours at 37° C. The cells arethen collected, suspended in PBS, fixed with 70% ice-cold methanol andstained with propidium iodide. The DNA content is then measured using aFACScan flow cytometer. Cell cycle phase distribution can then beestimated using CellFIT software (Becton-Dickinson).

HUV-EC-C Assay

This assay is used to measure a compound's activity against PDGF-R,FGF-R, VEGF, aFGF or Flk-1/KDR, all of which are naturally expressed byHUV-EC cells.

DAY 0

1. Wash and trypsinize HUV-EC-C cells (human umbilical vein endothelialcells, (American Type Culture Collection, catalogue no. 1730 CRL). Washwith Dulbecco's phosphate-buffered saline (D-PBS, obtained from GibcoBRL, catalogue no. 14190-029) 2 times at about 1 ml/10 cm² of tissueculture flask. Trypsinize with 0.05% trypsin-EDTA in non-enzymatic celldissociation solution (Sigma Chemical Company, catalogue no. C-1544).The 0.05% trypsin is made by diluting 0.25% trypsin/1 mM EDTA (Gibco,catalogue no. 25200-049) in the cell dissociation solution. Trypsinizewith about 1 ml/25-30 cm² of tissue culture flask for about 5 minutes at37° C. After cells have detached from the flask, add an equal volume ofassay medium and transfer to a 50 ml sterile centrifuge tube (FisherScientific, catalogue no. 05-539-6).

2. Wash the cells with about 35 ml assay medium in the 50 ml sterilecentrifuge tube by adding the assay medium, centrifuge for 10 minutes atapproximately 200× g, aspirate the supernatant, and resuspend with 35 mlD-PBS. Repeat the wash two more times with D-PBS, resuspend the cells inabout 1 ml assay medium/15 cm² of tissue culture flask. Assay mediumconsists of F12K medium (Gibco BRL, catalogue no. 21127-014) and 0.5%heat-inactivated fetal bovine serum. Count the cells with a CoulterCounter® (Coulter Electronics, Inc.) and add assay medium to the cellsto obtain a concentration of 0.8-1.0×10⁵ cells/ml.

3. Add cells to 96-well flat-bottom plates at 100 μl/well or 0.8-1.0×10⁴cells/well, incubate ˜24 h at 37° C., 5% CO₂.

Day 1

1. Make up two-fold test compound titrations in separate 96-well plates,generally 50 μM on down to 0 μM. Use the same assay medium as mentionedin day 0, step 2 above. Titrations are made by adding 90 μl/well of testcompound at 200 μM (4× the final well concentration) to the top well ofa particular plate column. Since the stock test compound is usually 20mM in DMSO, the 200 μM drug concentration contains 2% DMSO.

A diluent made up to 2% DMSO in assay medium (F12K+0.5% fetal bovineserum) is used as diluent for the test compound titrations in order todilute the test compound but keep the DMSO concentration constant. Addthis diluent to the remaining wells in the column at 60 μl/well. Take 60μl from the 120 μl of 200 μM test compound dilution in the top well ofthe column and mix with the 60 μl in the second well of the column. Take60 μl from this well and mix with the 60 μl in the third well of thecolumn, and so on until two-fold titrations are completed. When thenext-to-the-last well is mixed, take 60 μl of the 120 μl in this welland discard it. Leave the last well with 60 μl of DMSO/media diluent asa non-test compound-containing control. Make 9 columns of titrated testcompound, enough for triplicate wells each for: (1) VEGF (obtained fromPepro Tech Inc., catalogue no. 100-200, (2) endothelial cell growthfactor (ECGF) (also known as acidic fibroblast growth factor, or aFGF)(obtained from Boehringer Mannheim Biochemica, catalogue no. 1439 600),or, (3) human PDGF B/B (1276-956, Boehringer Mannheim, Germany) andassay media control. ECGF comes as a preparation with sodium heparin.

2. Transfer 50 μl/well of the test compound dilutions to the 96-wellassay plates containing the 0.8-1.0×10⁴ cells/100 μl/well of theHUV-EC-C cells from day 0 and incubate ˜2 h at 37° C., 5% CO₂.

3. In triplicate, add 50 μl/well of 80 μg/ml VEGF, 20 ng/ml ECGF, ormedia control to each test compound condition. As with the testcompounds, the growth factor concentrations are 4× the desired finalconcentration. Use the assay media from day 0 step 2 to make theconcentrations of growth factors. Incubate approximately 24 hours at 37°C., 5% CO₂. Each well will have 50 μl test compound dilution, 50 μlgrowth factor or media, and 100 μl cells, which calculates to 200μl/well total. Thus the 4× concentrations of test compound and growthfactors become 1X once everything has been added to the wells.

Day 2

1. Add ³H-thymidine (Amersham, catalogue no. TRK-686) at 1 μCi/well (10μl/well of 100 μCi/ml solution made up in RPMI media+10%heat-inactivated fetal bovine serum) and incubate ˜24 h at 37° C., 5%CO₂. RPMI is obtained from Gibco BRL, catalogue no. 11875-051.

Day 3

1. Freeze plates overnight at −20° C.

Day 4

Thaw plates and harvest with a 96-well plate harvester (Tomtec Harvester96®) onto filter mats (Wallac, catalogue no. 1205-401), read counts on aWallac Betaplate™ liquid scintillation counter.

In vivo Animal Models Assays

Xenograft Animal Models

The ability of human tumors to grow as xenografts in athymic mice (e.g.,Balb/c, nu/nu) provides a useful in vivo model for studying thebiological response to therapies for human tumors. Since the firstsuccessful xenotransplantation of human tumors into athymic mice,(Rygaard and Povlsen, 1969, Acta Pathol. Microbial. Scand. 77:758-760),many different human tumor cell lines (e.g., mammary, lung,genitourinary, gastro-intestinal, head and neck, glioblastoma, bone, andmalignant melanomas) have been transplanted and successfully grown innude mice. The following assays may be used to determine the level ofactivity, specificity and effect of the different compounds of thepresent invention. Three general types of assays are useful forevaluating compounds: cellular/catalytic, cellular/biological and invivo. The object of the cellular/catalytic assays is to determine theeffect of a compound on the ability of a TK to phosphorylate tyrosineson a known substrate in a cell. The object of the cellular/biologicalassays is to determine the effect of a compound on the biologicalresponse stimulated by a TK in a cell. The object of the in vivo assaysis to determine the effect of a compound in an animal model of aparticular disorder such as cancer.

Suitable cell lines for subcutaneous xenograft experiments include C6cells (glioma, ATCC #CCL 107), A375 cells (melanoma, ATCC #CRL 1619),A431 cells (epidermoid carcinoma, ATCC #CRL 1555), Calu 6 cells (lung,ATCC #HTB 56), PC3 cells (prostate, ATCC #CRL 1435), SKOV3TP5 cells andNIH 3T3 fibroblasts genetically engineered to overexpress EGFR, PDGFR,IGF-1R or any other test kinase. The following protocol can be used toperform xenograft experiments:

Female athymic mice (BALB/c, nu/nu) are obtained from SimonsenLaboratories (Gilroy, Calif.). All animals are maintained underclean-room conditions in Micro-isolator cages with Alpha-dri bedding.They receive sterile rodent chow and water ad libitum.

Cell lines are grown in appropriate medium (for example, MEM, DMEM,Ham's F10, or Ham's F12 plus 5%-10% fetal bovine serum (FBS) and 2 mMglutamine (GLN)). All cell culture media, glutamine, and fetal bovineserum are purchased from Gibco Life Technologies (Grand Island, N.Y.)unless otherwise specified. All cells are grown in a humid atmosphere of90-95% air and 5-10% CO₂ at 37° C. All cell lines are routinelysubcultured twice a week and are negative for mycoplasma as determinedby the Mycotect method (Gibco).

Cells are harvested at or near confluency with 0.05% Trypsin-EDTA andpelleted at 450× g for 10 min. Pellets are resuspended in sterile PBS ormedia (without FBS) to a particular concentration and the cells areimplanted into the hindflank of the mice (8-10 mice per group, 2-10×10⁶cells/animal). Tumor growth is measured over 3 to 6 weeks using veniercalipers. Tumor volumes are calculated as a product oflength×width×height unless otherwise indicated. P values are calculatedusing the Students t-test. Test compounds in 50-100 μL excipient (DMSO,or VPD:D5W) can be delivered by IP injection at different concentrationsgenerally starting at day one after implantation.

Tumor Invasion Model

The following tumor invasion model has been developed and may be usedfor the evaluation of therapeutic value and efficacy of the compoundsidentified to selectively inhibit KDR/FLK-1 receptor.

Procedure

8 week old nude mice (female) (Simonsen Inc.) are used as experimentalanimals. Implantation of tumor cells can be performed in a laminar flowhood. For anesthesia, Xylazine/Ketamine Cocktail (100 mg/kg ketamine and5 mg/kg Xylazine) are administered intraperitoneally. A midline incisionis done to expose the abdominal cavity (approximately 1.5 cm in length)to inject 10⁷ tumor cells in a volume of 100 μl medium. The cells areinjected either into the duodenal lobe of the pancreas or under theserosa of the colon. The peritoneum and muscles are closed with a 6-0silk continuous suture and the skin is closed by using wound clips.Animals are observed daily.

Analysis

After 2-6 weeks, depending on gross observations of the animals, themice are sacrificed, and the local tumor metastases to various organs(lung, liver, brain, stomach, spleen, heart, muscle) are excised andanalyzed (measurement of tumor size, grade of invasion, immunochemistry,in situ hybridization determination, etc.).

Additional Assays

Additional assays which may be used to evaluate the compounds of thisinvention include, without limitation, a bio-flk-1 assay, an EGFreceptor-HER2 chimeric receptor assay in whole cells, a bio-src assay, abio-lck assay and an assay measuring the phosphorylation function ofraf. The protocols for each of these assays may be found in U.S.application Ser. No. 09/099,842, which is incorporated by reference,including any drawings, herein.

B. Examples—Biological Activity.

Examples of the in vitro potency of compounds of this invention areshown in Table 2.

TABLE 2 bio bio bio FGFR1 flkGST EGFR bio PDGFR cdk2SPA IC50 IC50 IC50IC50 IC50 Example (mM) (mM) (mM) (mM) (mM) 1 0.22 0.05 2.54 2 4.880.12 >20 14.59 0.03 3 5.57 1.09 >20 >20 0.08 4 >20 0.009 >20 2.03 0.75 51.18 0.035 >20 7.57 6 1.6 0.009 >20 0.73 7 1.32 0.028 >20 0.73 8 7.016.07 >20 >20 9 11.04 1.14 >20 8.77 10 1.25 0.07 >20 0.39 11 10.911.41 >20 9.29 12 1.1 0.49 16.33 5.47 14 0.03 0.06 17.95 14.3715 >20 >20 >20 >20 16 5.2 7.91 >20 1.02 17 2.08 0.57 >20 1.74 18 0.080.02 >20 0.39 19 0.87 0.19 >20 8.61 29 3.83 0.71 >20 1.88 0.01 30 1.340.07 >20 1.23 0.02 31 0.98 0.06 >20 1.5 0.02 32 2.09 0.51 >20 1.21 0.00933 0.06 34 0.26 35 1.65 0.14 36 3.78 0.03 37 1.31 0.12 38 2.18 0.18 >2039 16.4 1.24 40 5.26 0.32 >20 1.7 41 13.52 5.94 >20 >20 42 0.31 0.17 >201.78 43 0.24 0.07 >20 0.69 44 0.05 0.03 19.13 0.4 1.9645 >20 >20 >20 >20 46 19.09 5.03 >20 >20 47 1.1 0.24 >20 3.28 48 0.260.02 >20 0.15 49 3.7 14.64 >20 4.63 50 2.81 4.41 14.74 4.89 51 0.220.01 >20 2.31 52 10.09 10.5 >20 19.05 53 2.24 1.51 >20 13.3854 >20 >20 >20 >20 55 2.08 1.44 >20 3.84 60 8.51 1.97 >20 10.38 61 4.570.62 >20 12.54 62 0.92 0.07 >20 1.47 63 1.22 0.21 >20 6.58 64 1.190.45 >20 4.57 65 2.55 0.78 >20 14.36 66 0.19 0.018 >20 2.1 67 0.530.036 >20 1.68 68 1.18 0.16 >20 3.37 69 1.08 0.02 >20 2.04 70 19.953.1 >20 5.6 71 2.19 0.43 >20 7.51 72 1.23 0.008 >20 1.77

7. Measurement of Cell Toxicity

Therapeutic compounds should be more potent in inhibiting receptortyrosine kinase activity than in exerting a cytotoxic effect. A measureof the effectiveness and cell toxicity of a compound can be obtained bydetermining the therapeutic index, i.e., IC₅₀/LD₅₀. IC₅₀, the doserequired to achieve 50% inhibition, can be measured using standardtechniques such as those described herein. LD₅₀, the dosage whichresults in 50% toxicity, can also be measured by standard techniques aswell (Mossman, 1983, J. Immunol. Methods, 65:55-63), by measuring theamount of LDH released (Korzeniewski and Callewaert, 1983, J. Immunol.Methods, 64:313, Decker and Lohmann-Matthes, 1988, J. Immunol. Methods,115:61), or by measuring the lethal dose in animal models. Compoundswith a large therapeutic index are preferred. The therapeutic indexshould be greater than 2, preferably at least 10, more preferably atleast 50.

Conclusion

It would be appreciated that the compounds, methods and pharmaceuticalcompositions of the present invention are effective in modulating PKactivity and therefore are expected to be effective as therapeuticagents against RTK, CTK-, and STK-related disorders.

One skilled in the art would also readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent herein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described.

Other embodiments are within the following claims.

What is claimed:
 1. A compound of the formula:

wherein: R¹ and R² are independently selected from the group consistingof hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo,—CX₃, hydroxy, alkoxy, nitro, cyano, —C(O)R26, —C(O)OR²⁶, R²⁶C(O)O—,—C(O)NR²⁸R²⁹, R²⁶C(O)NR²⁸—, —NR²⁸R²⁹, —S(O)2R²⁶, —S(O)₂OR²⁶,—S(O)₂NR²⁸R²⁹, R²⁶S(O)₂NR²⁸—, X₃CS(O)2— and X₃CS(O)₂NR²⁸— where X is F,Cl, Br, or I;

wherein: D is carbon or nitrogen; R⁸, R⁹, R¹¹ and R¹² are independentlyselected from the group consisting of hydrogen, alkyl, hydroxy, alkoxy,halo, nitro, cyano and —NR²⁸R²⁹; Z is selected from the group consistingof oxygen, sulfur, and —NR¹⁰; R¹⁰ is selected from the group consistingof hydrogen, alkyl, cycloalkyl, aryl, —C(O)R²⁶, —C(S)R²⁶, —C(O)R²⁶,—C(O)NR²⁸R²⁹, —C(S)NR²⁸R²⁹, —C(NH)NR²⁸R²⁹ and —S(O)2R²⁶; Q is selectedfrom the group consisting of:

where: G¹, G², G³, G⁴ and G⁵ are selected from the group consisting ofcarbon and nitrogen with the proviso that no more than two of G¹, G²,G³, G⁴ and G⁵ are nitrogen; R¹⁷, R⁸, R¹⁹, R²⁰ and R²¹ are independentlyselected from the group consisting of hydrogen, alkyl, hydroxy, alkoxy,halo, —NR²⁸R²⁹,—(CH₂)_(n)C(O)R²⁶, —(CH₂)_(n)C(O)OR²⁶ and—(CH₂)_(n)C(O)NR²⁸R²⁹, —(CH₂)_(n)NR²⁸R²⁹, —(CH₂)_(n)S(O)₂R²⁶ and—CH₂)_(n)S(O)₂NR²⁸R²⁹; J¹ is selected from the group consisting ofnitrogen, oxygen and sulfur such that when J¹ is nitrogen, R²² isselected from the group consisting of hydrogen, alkyl and —C(O)R²⁶; andwhen J¹ is oxygen or sulfur, R²² does not exist; J², J³ and J⁴ areselected from the group consisting of carbon and nitrogen; R²³, R²⁴ andR²⁵ are independently selected from the group consisting of hydrogen,alkyl, aryl optionally substituted with one or more groups independentlyselected from the group consisting of hydroxy, unsubstituted loweralkoxy and halo, halo, —(CH₂)_(n)C(O)R²⁶, —(CH₂)_(n)C(O)OR²⁶ and—CH₂)_(n)C(O)NR²⁸R²⁹, —(CH₂)_(n)NR²⁸R²⁹, —(CH₂)_(n)S(O)₂R^(26, —(CH)₂)_(n)S(O)₂NR²⁸R²⁹, —(CH₂)_(n)OR²⁶, —O(CH₂)_(n)NR²⁸R²⁹ and—C(O)NH(CH₂)_(n)NR²⁸R²⁹; n is 0, 1, 2, or 3; R²³ and R²⁴ or R²⁴ and R²⁵may combine to form a group selected from the group consisting of—CH₂CH₂CH₂CH₂—, —CH═CR³³CR³⁴═CH— and —C(O)Y(CH₂)₂— and group wherein Yis selected from the group consisting of oxygen, sulfur and N(R²⁷)— andR³³ and R³⁴ are selected from the group consisting of hydrogen,—(CH₂)_(n)NR²⁸R²⁹ and —O(CH₂)_(n)NR²⁸R²⁹ where, when one of R³³ or R³⁴is —(CH₂)_(n)NR²⁸R²⁹ or —O(CH₂)_(n)NR²⁸R²⁹, the other is hydrogen; itbeing understood that, when J², J³ or J⁴ is nitrogen, R²³, R²⁴ or R²⁵,respectively, does not exist; R²⁶ is selected from the group consistingof hydrogen, alkyl, cycloalkyl, aryl and heteroaryl; R²⁷ is selectedfrom the group consisting of hydrogen and alkyl; R²⁸ and R²⁹ areindependently selected from the group consisting of hydrogen, alkyl,aryl, heteroaryl, —(CH₂)_(n)aryl, —(CH₂)_(n)heteroaryl and —C(O)R²⁶, or,combined, R²⁸ and R²⁹ may form a group selected from the groupconsisting of —(CH₂)₅—, —(CH₂)₂O(CH₂)₂—, —CH₂)₂NR³⁰(CH₂)₂— and—(CH)₃C(O)— wherein R³⁰ is selected from the group consisting ofhydrogen, alkyl, —C(O)R²⁶, —S(O)₂R²⁶, —S(O)₃R²⁶, —S(O)₂NR³¹R³²,—C(O)NHNR³¹R³², —C(O)NR³¹R³², —C(S)NR³¹R³² and —C(O)₀R²⁶ where R³¹ andR³² are independently selected from the group consisting of hydrogen,unsubstituted lower alkyl and aryl optionally substituted with one ormore groups independently selected from the group consisting of halo andunsubstituted lower alkoxy; or a pharmaceutically acceptable saltthereof.
 2. The compound of claim 1, wherein R¹ and R² are hydrogen. 3.The compound of claim 1, wherein Het is:

wherein: D is carbon; R⁸, R⁹, R¹¹ and R¹² are hydrogen; and Z is —NR¹⁰where R¹⁰ is selected from the group consisting of —C(O)R²⁶, —C(O)OR²⁶,—C(O)NR²⁸R²⁹, —C(S)NR²⁸R²⁹ and —C(NH)NR²⁸R²⁹.
 4. The compound of claim3, wherein Het is piperidin—4—yl.
 5. The compound of claim 1, wherein Qis:

wherein: J¹ is nitrogen; J², J³ and J⁴ are carbon; and R²² is hydrogen.6. The compound of claim 5, wherein: R²³ is selected from the groupconsisting of hydrogen, unsubstituted lower alkyl, —C(O)OR²⁶,—C(O)NR²⁸R²⁹ or R²³ combined with R²⁴ form —(CH₂)₅— and —CH═CH—CR³⁴═CH—where R²⁶ is hydrogen or unsubstituted lower alkyl; R³⁴ is selected fromthe group consisting of hydrogen and —O(CH₂)NR²⁸R²⁹ and R²⁸ and R²⁹ areindependently selected from the group consisting of hydrogen,unsubstituted lower alkyl and, R²⁸ and R²⁹ combined, form a groupselected from the group consisting of —(CH₂)₂N(R³⁰)(CH₂)₂—,—(CH₂)₂O(CH₂)₂— and —(CH₂)₅—, wherein R³⁰ is selected from the groupconsisting of hydrogen and unsubstituted lower alkyl.
 7. The compound ofclaim 6, wherein R²⁴ and R²⁵ are independently selected from the groupconsisting of: hydrogen; unsubstituted lower alkyl; aryl optionallysubstituted with a group selected from the group consisting of halo,unsubstituted lower alkoxy; morpholino and 4—formylpiperidinyl;—(CH₂)_(n)C(O)NR²⁸R²⁹; —(CH₂)_(n)C(O)OR²⁶; —(CH₂)_(n)NR²⁸R²⁹;—(CH₂)_(n)OR²⁶, —C(O)NH(CH₂)_(n)NR²⁸R²⁹; —O(CH₂)_(n)NR²⁸R²⁹;—O(CH₂)_(n)OR²⁶, and, when R²⁴ is not combined with R²³, R²⁴ an R²⁵combined form a group selected from the group consisting of:—(CH₂)₂OC(O)—; —(CH₂)₂N(R³⁰)C(O)—; —(CH₂)₅—; and —CH═CH—CH═CH—; whereR²⁶ is selected from the group consisting of hydrogen and unsubstitutedlower alkyl; R²⁸ and R²⁹ are independently selected from the groupconsisting of hydrogen, unsubstituted lower alkyl, lower alkylsubstituted with a phenyl or a pyridyl group or, combined, a groupselected from the group consisting of —(CH₂)₅—, —(CH₂)₂NR³⁰(CH₂)₂— and—(CH₂)₂O(CH₂)₂— where R³⁰ is selected from the group consisting ofhydrogen, unsubstituted lower alkyl and —C(O)R²⁶ where R²⁶ is as definedabove.
 8. The compound of claim 1, wherein Q is3,5-dimethyl-4-(4-methylpiperazin-1-yl-carbonyl)-1H-pyrrol-2-yl,5-(methyl-3H-imidazol-4-yl)-1H-pyrrol-2-yl,3-methyl-4-(4-methylpiperidin-1-yl-carbonyl)-1H-pyrrol-2-yl,3,5-dimethyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4,5,6,7-tetrahydro-1H-indol-2-yl,3-(2-carboxyethyl)-5-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-5-ethyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4-ethoxycarbonyl-5-methyl-1H-pyrrol-2-yl,4-(2-carboxyethyl)-3,5-dimethyl-1H-pyrrol-2-yl,4-(carboxymethyl)-3,5-dimethyl-1H-pyrrol-2-yl, indol-2-yl,4,5,6,7-tetrahydroindol-2-yl, 5-(2-morpholin-4-ylethyloxy)indol-2-yl,3-(carboxy)-5-methyl-1H-pyrrol-2-yl, 5-carboxy-3-methyl-1H-pyrrol-2-yl,3-(3-morpholin-4-ylpropyl)-4,5,6,7-tetrahydroindol-2-yl,4-(2-diethylaminoethylaminocarbonyl)-3,5-dimethyl-1H-pyrrol-2-yl,4-(4-methylpiperazin-1-ylcarbonyl)-3,5-dimethyl-1H-pyrrol-2-yl,5-(4-methylpiperazin-1-ylcarbonyl)-3-methyl-1H-pyrrol-2-yl,5-(ethoxycarbonyl)-4,5,6,7-tetrahydro-2H-isoindol-3-yl,4-(pyridin-4-ylaminocarbonyl)-3-phenyl-5-methyl-1H-pyrrol-2-yl,5-methylthiophen-2-yl,3-(2-carboxyethyl)-5-ethoxycarbonyl-4-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4-carboxy-1H-pyrrol-2-yl,3-(4-hydroxyphenyl)-4-ethoxycarbonyl-1H-pyrrol-2-yl,4-(morpholin-4-ylcarbonyl)-3-methyl-1H-pyrrol-2-yl,4-(piperidin-1-ylcarbonyl)-3-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-5-(ethoxycarbonyl)-4-methyl-1H-pyrrol-2-yl,3-(2-carboxyethyl)-4-(carboxy)-1H-pyrrol-2-yl,3-(methyl)-4-(benzylaminocarbonyl)-1H-pyrrol-2-yl,3-methyl-4-(pyridin-4-ylmethylaminocarbonyl)-1H-pyrrol-2-yl,3-methyl-4-[3-(2-oxopyrrolidin-1-yl)propyl-aminocarbonyl)-1H-pyrrol-2-yl,5-methyl-4-ethoxycarbonyl-3-[3-(4-methylpiperazin-1-yl)propyl]-1H-pyrrol-2-yl,or 3,5-dimethyl-4-(4-methylpiperazin-1-ylaminocarbonyl)-1H-pyrrol-2-yl.9. The compound of claim 8, wherein R¹ and R² are hydrogen.
 10. Thecompound of claim 9, wherein Het is piperidin—4—yl.
 11. The compound ofclaim 1, wherein Q is selected from the group consisting of:


12. A pharmaceutical composition comprising a compound or salt of claim1 and a pharmaceutically acceptable carrier or excipient.
 13. Apharmaceutical composition comprising a compound or salt of claim 10 anda pharmaceutically acceptable carrier or excipient.